Methods and uses of leptin in hepatocellular carcinoma

ABSTRACT

The present invention relates to methods for the modulation of immune responses. More particularly, the invention relates to methods and uses of leptin for immuno-modulation of the balance between the Th1/Th2 responses and for the treatment of immune-related disorders. Specifically, the methods of the invention comprise shifting the Th1/Th2 cell balance towards the proinflammatory state. This modulation of the cell balance may be performed by increasing the activity and/or the expression of leptin in said subject, for instance by raising the amount of leptin. The amount of leptin in a subject may be increased by activating immunoregulatory cells, administering an immunomodulatory amount of leptin or a homologue, derivative, or functional fragment of leptin.

FIELD OF THE INVENTION

The present invention relates to methods for modulation of immuneresponse. More particularly, the invention relates to methods and usesof leptin for immuno-modulation of the balance between Th1-Th2 responsesand for the treatment of immune-related disorders.

BACKGROUND OF THE INVENTION

The immune system plays a major part in tumor pathogenesis. In animalmodels and in humans it has been demonstrated that tumor cell formationleads to T cell activation aimed at tumor cell destruction, while thedevelopment of cancer is related to specific tumor-specific anergy.Conditions of immunodeficiency, such as infection with HIV and prolongedimmunosuppressive therapy are associated with an increased incidence ofcancer formation. Current anti-cancer immune therapies are aimed atcorrecting immune derangements in malignancy-harboring hosts. Severalexperimental and clinical immunomodulatory strategies are employed toactivate an anti-tumor pro-inflammatory immune response in systemicallydisseminated tumors. Examples are numerous and include IL2 and IFN-αtreatment in metastatic melanoma [Eigentler, T. K. et al., Lancet Oncol.4(12):748-59 (2003)], IFN treatment in metastatic renal cell carcinoma[Gitlitz, B. J. and Figlin, R. A. Urol. Clin. North. Am. 30(3):589-600(2003)], TNF-α therapy in metastatic thyroid carcinoma [Mitsiades, C. S.et al., Endocrinol. 178(2):205-16 (2003)], the use ofgranulocyte-colony-stimulating-factor (GCSF) as a dendritic cellactivator in breast, prostate, and renal cancer [Waller, E. K. andErnstoff, M. S. Cancer 1; 97(7):1797-809 (2003)], and transfer ofalloreactive donor T lymphocytes to promote the ‘graft versus leukemia’effect in hematological malignancies [Costello, R. T., et al., Eur. J.Haematol. 70(5):333-45 (2003)].

Leptin is an 16 kDa product of the ob gene that was discovered in 1994[Zhang, Y. et al., Nature 372:425-432 (1994)]. This protein is secretedalmost exclusively by adipose cells, and acts centrally at thehypothalamic region in regulating energy expenditure and appetite[Pelleyrnounter, M. A. et al., Science 269:540-543 (1995)]. Leptindeficient ob/ob mice suffer from morbid obesity, diabetes mellitus,hyperlipidemia and hepatic steatosis, while leptin administration tothese mice results in reversal of these disorders [Halaas, J. L. etal.,. Science 269:543-546 (1995)]. In contrast, the administration ofleptin to humans suffering from morbid obesity failed to suppressappetite or reduce weight [Friedman, J. M. et al., Nutr. Rev. 60(10 Pt2):S1-14; discussion S68-84, 85-7 (2002)]. This disappointing result maystem from increased leptin levels in obese individuals, and ‘leptinresistance’ caused by other, newly discovered, regulatory proteins suchas adiponectin [Mark, A. L. et al., J. Hypertens. 20(7):1245-50 (2002);Berg, A. I {. et aT., Trends Endocrinol. Metab. 13(2):84-9 (2002)].

It has been previously suggested that leptin possesses potentimmunomodulatory properties [Loffreda, S. et al, 12(1):57-65 (1998)].Structurally, leptin is similar to IL2, IL6, and IL15, making it amember of the helical cytokine superfamily [Madej, T. et al., FEBS Lett.2;373(1):13-8 (1995)], while leptin receptors are structurally similarto hematopoietic cytokine receptors [Gimble, J. M. et al., Bone19(5):421-8 (1996)]. Leptin receptors are found on CD4 and CD8lymphocytes, monocytes [Sanchez-Margalet, V. Clin. Exp. Immunol.129(1):119-24 (2002)], natural-killer lymphocytes [Zhao, Y. et al.,Biochem. Biophys. Res. Commun. 10:300(2):247-52 (2003); Motivala, S. J.et at., Alcohol. Clin. Exp. Res. 27(11):1819-24 (2003)], and hepaticstellate cells [Saxena, N. K. et al., Hepatology 35(4):762-71 (2002)].Leptin enhances T cell proliferation and pro-inflammatory cytokinesecretion [Lord, G. M. et al., Nature 27;394(6696):897-901 (1998); Lord,G. M. et al., J. Leukoc. Biol. 72(2):330-8 (2002); Martin-Romero, C. etal., Cell. Immunol. 10;199(1):15-24 (2000)], by activating the JAK/STATsignal transduction pathway [Cao, Q. et al., J. Biol. Chem.6;279(6):4292-304 (2004)]. Leptin deficient ob/ob mice are immunedeficient, suffering from an increased propensity for infection andmortality [Faggioni, R. et at., FASEB J. 15(14):2565-71 (2001)] andimpaired function of NKT lymphocytes and hepatic macrophages [Li, Z. etal., Gastroenterology 123(4):1304-10 (2002)]. These mice are resistantto several Th1 mediated immune disorders, including allergicexperimental encephalomyelitis [Matarese, G. et al., J. Immunol.15;166(10):5909-16 (2001)], concanavalin A hepatitis [Siegmund, B. etal., Eur. J. Immunol. 32(2):552-60 (2002)], experimental arthritis[Busso, N. et al., J. Immunol. 15;168(2):875-82 (2002)], and autoimmunenephritis [Tarzi, R. M. et al., Am. J. Pathol. 164(2):385-90 (2004)] butare extremely vulnerable to LPS-induced hepatic damage [Yang, S. et al.,Am. J. Physiol. Gastrointest. Liver Physiol. 281(2):G382-92 (2001)].Leptin replenishment reverses all of these effects.

Wildtype mice that do not suffer from leptin deficiency develop apotentiation in severity of experimental allergic encephalomyelitis[Matarese, G. et al., Eur. J. Immunol. 31(5):1324-32 (2001)] and type Idiabetes mellitus [Matarese, G. et al. Diabetes 51(5):1356-61. (2002)].In humans, no trials have examined the relationship between leptin andimmune phenomena. However, the elevated leptin levels among females(that also suffer from an unexplained increased tendency for thedevelopment of autoimmune disease) and the reduced levels of leptinamong malnourished individuals (that also tend to develop secondaryimmune deficiency) may point out to leptin being a link betweennutrition and immunity [Matarese, G. et al., Trends in immunology.23(4):182-7 (2002)].

Aberrant leptin receptors have been found on breast [Hu, X. et al., J.Natl. Cancer. Inst. 20;94(22):1704-11 (2002)], endometrial [Yuan, S. S.et al., Gynecol. Oncol. 92(3):769-75 (2004)], bladder [Yuan, S. S. etal., Urology. 63(2):408-13 (2004)], and esophageal [Somasundar, P. etal., Am. J. Surg. 186(5):575-8 (2003)] cancer cell lines. Leptin hasbeen recently demonstrated to activate proliferation of breast[Catalano, S. et al., S. Biol. Chem. 25 (2004)], prostate [Onuma, M. etal., J. Biol. Chem. 24:278(43):42660-7 (2003)], and colon[Rouet-Benzineb, P. et al., S. Blot. Chem. 29 (2004)] cancer cell linesthrough direct activation of the JAK/STAT signal transduction pathway.In contrast, leptin was shown to inhibit proliferation of pancreatic[Somasundar P, McFadden D W, Hileman S M, Vona-Davis L. Leptin is agrowth factor in cancer. J Surg Res. 2004; 116(2):337-49] and colon[Aparicio T, Guilmeau S, Goiot H, Tsocas A, Laigneau J P, Bado A,sobhani I, Lehy T. Leptin reduces the development of the initialprecancerous lesions induced by azoxymethane in the rat colonic mucosa.Gastroenterology. 2004; 126(2):499-510]. While leptin levels were shownto be low in patients with gastrointestinal [Dulger H, Alici S,Sekeroglu M R, Erkog R, Ozbek H, Noyan T, Yavuz M. Serum levels ofleptin and proinflammatory cytokines in patients with gastrointestinalcancer. Int J Clin Pract. 2004;58(6):545-9] [Bolukbas F F, Kilic H,Bolukbas C, Gumus M, Horoz M, Turhal N S, Kavakli B. Serum leptinconcentration and advanced gastrointestinal cancers: a case controlledstudy. BMC Cancer. 2004;4(1):29] and pancreatic malignancies [Barber MD, McMillan D C, Wallace A M, Ross J A, Preston T, Fearon K C. Theresponse of leptin, interleukin-6 and fat oxidation to feeding inweight-losing patients with pancreatic cancer. Br J Cancer. 2004;90(6):1129-32], they were normal in patients with breast cancer [Stattin P,Soderberg S, Biessy C, Lenner P, Hallmans G, Kaaks R, Olsson T. Plasmaleptin and breast cancer risk: a prospective study in northern Sweden.Breast Cancer Res Treat. 2004;86(3):191-6], high in patients withcolorectral cancer [Stattin P, Palmqvist R, Soderberg S, Biessy C,Ardnor B, Hallmans G, Kaaks R, Olsson T. Plasma leptin and colorectalcancer risk: a prospective study in Northern Sweden. Oncol Rep.2003;10(6):2015-21], and variable in patients receiving chemotherapy forhematological malignancy [Minami R, Muta K, Ilseung C, Matsushima T, AbeY, Nishimura J, Nawata H. Plasma leptin levels vary with the periodsduring chemotherapy. Am J Hematol. 2003; 74(2): 145]. Leptin may exerttwo opposing effects—a direct, pro-proliferative effect on tumor cellsand an indirect, pro-inflammatory and anti-tumoral immune response. Thenet balance between these two leptin-induced reactions may determinewhether it may play a pro or anti tumorogenic effect.

The present invention shows a clear involvement of leptin in modutationof Th1 immune response, and pro-inflammatory cytokine secretion,particularly through NKT lymphocyte activation. Such modulation enablesthe use of leptin as an immunomodulator for the treatment of differentimmune-related disorders, particularly, disorders involving NK cells.

It is therefore an object of the invention to provide a method for themodulation of Th1-Th2 response, and more particularly to enhance apro-inflammatory cytokine secretion.

Another object of the invention is to provide method for the treatmentimmune-related disorders by modulating the expression of leptin.

The invention further provides for the use of leptin in the treatment ofimmune-related disorders and in immuno-modulation.

These and other objects of the invention will become apparent as thedescription proceeds.

SUMMARY OF THE INVENTION

In a first aspect, the present invention relates to a method forimmuno-modulation of the Th1/Th2 cell balance, in a subject in needthereof, comprising the step of modulating the levels, the expressionand/or the activity of leptin in said subject.

According to one embodiment, modulation of the Th1/Th2 cell balance bythe method of the invention may be performed by increasing or decreasingthe levels, the activity and/or the expression of leptin in saidsubject.

According to another preferred embodiment, modulation of the Th1/Th2cell balance may be mediated by the activation of immuno-regulatorycells selected from the group consisting of NK T cells, antigenpresenting cells and CD4⁺, CD25⁺ T cells, and preferably, NK T cells, byleptin.

In one specific embodiment, the invention provides for a method forimmuno-modutation resulted by the shifting of the Th1/Th2 cell balancetoward the pro-inflammatory cytokine producing cells, in a subject inneed thereof. Accordingly, this method comprises the step of increasingthe levels, the expression and/or the activity of leptin in saidsubject.

More particularly, increasing the amount of leptin, the expression ofleptin or leptin activity may be by carried out by a variety of means.For instance proteins or nucleic acids coding for proteins may beadministered to the subject. The most obvious example of such a proteinwould be leptin itself, but it is also a subject of the presentinvention that homologues of leptin, functional derivatives of leptin orfragments of leptin may also be used if they are capable of inducingappropriate immune modulations. Another example of a protein is ananti-idiotypic antibody to leptin, since it has been previously shownthat an anti-idiotypic antibody is capable of inducing the sameinteractions with the leptin receptor that can be seen with the leptinmolecule itself [De Fanti et al., Obesity Research 10:833-837 (2002)].Rather than using these proteins themselves, nucleic acids may be usedthat code for these proteins after introduction into a cell. Forinstance, it has been previously shown that nucleic acid constructs thatexpress the leptin gene can induce cells to excrete leptin [MacDougaldet al., Proc Nat. Acad Sci USA 92:9034-9037 (1995)]. As describedpreviously constructs that express modified versions or portions of theleptin gene may also be used. Furthermore, there are small moleculesthat have been described in the literature among whose propertiesinclude the ability of raising the level or activity of leptin in asubject. For instance, thiazolidinedione, a drug that is used to treatdiabetes, has the effect of raising leptin levels but it has beendisclosed in U.S. patent application Ser. No. 20020032225 thatdiphenylethylene compounds that contain either thiazolidinedionemoieties or a related drug, oxazolidinedione have the opposite effectand raises leptin levels. Another example is nicotinic acid andnicotinic acid esters which have also been shown to increase the amountof leptin in a subject [U.S. patent application Ser. No. 20020128298].It should be noted that it is a subject of the present invention thatthese reagents may be used alone or they may be used in variouscombinations.

According to a specifically preferred embodiment, the method of theinvention is particularly useful for shifting of the Th1/Th2 cellbalance toward the pro-inflammatory cytokine producing cells a mammaliansubject suffering of an immune-related disorder.

More preferably, shifting of the Th1/Th2 cell balance toward thepro-inflammatory cytokine producing cells may be particularlyadvantageous for immune-related disorder such as malignant proliferativedisorder, a disorder caused by immuno-suppression or an infection causedby a pathogenic agent.

According to one embodiment, a malignant proliferative disorder may beany one of solid and non-solid tumors selected from the group consistingof carcinoma, sarcoma, melanoma, leukemia and lymphoma.

According to another particular embodiment, the method of the inventionmay be advantageous for Hepatocellular carcinoma (HCC).

In another preferred embodiment, the subject in need of modulating theTh1/Th2 cell balance toward the pro-inflammatory cytokine producingcells by the method of the invention may suffer from anyimmuno-suppressive disorder, for example immune suppression caused byinfection of an immunodeficiency virus, preferably, HIV, oralternatively, immuno-suppression caused by chemotherapy.

According to another embodiment, shifting of the Th1/Th2 cell balancetoward the pro-inflammatory cytokine producing cells by the method ofthe invention may be advantageous in a subject suffering of aninfection, for example infection caused by a pathogenic agent selectedfrom the group consisting of bacterial pathogens, viruses, fungi,parasites and yeast.

The invention further provides for a method for the treatment of animmune-related disorder in a subject in need thereof, comprising thestep of administering to said subject an immuno-modulatory effectiveamount of a protein or nucleic acid that will increase the amount ofleptin, the expression of leptin or leptin activity in the subject.Examples of proteins can comprise but not be limited to leptin,homologues of leptin, functional derivatives of leptin, leptin fragmentsand anti-idiotypic antibodies to leptins. Nucleic acids useful for thepurpose of increasing the amount, expression or activity of leptin canbe designed to express any of the foregoing proteins. Small moleculessuch as nicotinic acids, nicotinic acid ester or diphenylethylenecompounds that contain either thiazolidinedione or oxazolidinedionemoieties may also find use in the present invention. These compounds orreagents may be used alone or they maybe used in combination with eachother.

In one embodiment, the effective amount administered to a subjectsuffering from an immune-related disorder may be an amount sufficientfor shifting the Th1/Th2 cell balance toward pro-inflammatory cytokineproducing cells.

According to another embodiment, the method of the invention isparticularly applicable for the treatment of an immune-related disordersuch as any one of a malignant proliferative disorder, a disorder causedby immuno-suppression or an infection caused by a pathogenic agent.

More particularly, the method of the invention is particularlyapplicable for the treatment of a malignant proliferative disorder suchas any one of solid and non-solid tumors selected from the groupconsisting of carcinoma, sarcoma, melanoma, leukemia and lymphoma.

According to a specifically preferred embodiment, the method of theinvention is for the treatment of Hepatocellular carcinoma (HCC).

According to another embodiment, the method of the invention is suitablefor the treatment of immuno-suppression caused by infection of animmunodeficiency virus, preferably, HIV, or caused by chemotherapy.

Still further, the method of the invention is intended for the treatmentof an infection caused by a pathogenic agent, for example, bacterialpathogens, viruses, fungi, parasites or yeast.

In another aspect, the invention relates to the use of a protein ornucleic acid that increases the amount, expression or activity of leptinfor the preparation of a composition for shifting the Th1/Th2 cellbalance toward pro-inflammatory cytokine producing cells.

The invention further relates to the use of protein or nucleic acid thatincrease the amount, expression or activity of leptin for thepreparation of a medicament for the treatment of an immune-relateddisorder.

According to one embodiment of said aspect, the immune-related disordermay be any one of a malignant proliferative disorder, a disorder causedby immuno-suppression or an infection caused by a pathogenic agent.

Specifically, a malignant proliferative disorder may be any one of solidand non-solid tumors selected from the group consisting of carcinoma,preferably, Hepatocellular carcinoma (HCC), sarcoma, melanoma, leukemiaand lymphoma.

In another embodiment, the immune related disorder may beimmuno-suppression, for example, caused by infection of animmunodeficiency virus, preferably, HIV, or alternatively, bychemotherapy.

In yet another embodiment, the immune related disorder may be infectioncaused by a pathogenic agent selected from the group consisting ofbacterial pathogens, viruses, fungi, parasites and yeast.

In a third aspect, the invention relates to a method forimmuno-modulation of the immune system resulted by shifting the Th1/Th2cell balance toward the anti-inflammatory cytokine producing cells, in asubject in need thereof. Such method comprises the step of decreasingthe levels, the expression and/or the activity of leptin in saidsubject.

Decreasing the amount of leptin, the expression of leptin or theactivity of leptin can be carried out by a variety of means. Forinstance, an immunomodulatory effective amount of proteins or nucleicacids may be administered to the subject for this purpose. Examples ofproteins can comprise but not be limited to antibodies to either leptinor the leptin receptor which are obtainable from a wide variety ofcommercial sources. Competitors such as soluble leptin receptor [Yang etal., Molec Endricrinol 18:1354-1362 (2004)] or a fragment of Helix IIIof leptin [Gonzalez and Leavis, 2003 Endocrine 21:185-196 (2003)] canalso result in a loss of leptin signaling. Thus it is also a subject ofthe present invention that fragments of the leptin or the leptinreceptor may also find use with present invention. Alternatively,instead of the proteins themselves, nucleic acid constructs coding forthe foregoing proteins may also be used. Furthermore, nucleic acids maybe used which comprise sequences complementary to either leptin or theleptin receptor where the nucleic acids comprise antisense DNA,antisense RNA, ribozymes or small interfering RNA (siRNA) that targetleptin or the leptin receptor or they may be expression constructs thatdirect synthesis of antisense RNA, ribozymes or siRNA. In addition tothese means, other molecules may also be used that decrease the amountof leptin, the expression of leptin or the activity of leptin. Examplesof these can comprise but not be limited to thiazolidinediones [Kallenand Lazar, Proc. Nat, Acad. Sci. USA 93:5793-5796 (1996)], galanin [Liet al., J Mol Endocrinol. 33:11-19 (2004)], SOCS-3 (U.S. patentapplication 20040087530) and the beta 3-adrenoceptor agonists BRL35135Aand ZD2079 [Trayhurn et al., Biochem Biophys Res Commun 228:605-610(1996)]. These compounds or reagents may be used alone or they maybeused in combination with each other.

According to a specific embodiment, a subject may be a mammalian subjectsuffering from an immune-related disorder.

More particularly, the immune-related disorder may be any one of aninflammatory disorder, an autoimmune-disorder, a graft-rejectionassociated disorder or a fibrotic disorder.

In one embodiment, an inflammatory disorder may be an intestinalinflammatory disease, preferably, an inflammatory bowel diseases (IBD).

In another embodiment, the immune-related disorder may be a fibroticdisorder, for example, hepatic fibrosis, cardiac fibrosis, or colonfibrosis.

In yet another embodiment, shifting the Th1/Th2 cell balance toward theanti-inflammatory cytokine producing cells, may be performed in asubject suffering from an auto-immune disease such as arthritis,diabetes or immune related infertility disorders.

The invention further provides for a method for the treatment of animmune-related disorder in a subject in need thereof. The method of theinvention comprises the step of administering to said subject animmuno-modulatory effective amount of proteins or nucleic acids thatwill decrease the amount of leptin, the expression of leptin or leptinactivity. Examples of proteins can comprise but not be limited to leptinfragments, antibodies to leptin, fragments of antibodies to leptin,soluble leptin receptor, fragments of the leptin receptor, antibodies tothe leptin receptor and fragments of antibodies to leptin receptor,galanin and the SOCS-3 gene product. Examples of nucleic acids cancomprise but not be limited to oligonucleotides or nucleic acids thatact as antisense, ribozymes or siRNA or that code for expression ofantisense RNA, ribozymes or siRNA. These compounds or reagents may beused alone or they maybe used in combination with each other.

According to one embodiment, an effective amount may be an amountsufficient for shifting the Th1/Th2 cell balance towardanti-inflammatory cytokine producing cells.

In another embodiment, the method of the invention is intended for thetreatment of an immune-related disorder such as inflammatory disorder,an autoimmune-disorder a graft-rejection associated disorder or afibrotic disorder.

According to a specific embodiment, the method of the invention isparticularly advantageous for the treatment of an inflammatory disorder,preferably, an intestinal inflammatory disease or most preferably,inflammatory bowel diseases (IBD).

According to another specific embodiment, the method of the invention issuitable for the treatment of a fibrotic disorder such as any one ofhepatic fibrosis, cardiac fibrosis or colon fibrosis.

Still further, the method of the invention may be applicable for thetreatment of auto-immune disease, for example, any one of arthritis,diabetes or immune-related infertility disorders.

According to another aspect, the invention relates to the use of aprotein or nucleic acid that can reduce the amount, expression oractivity of leptin for the preparation of a composition for shifting theTh1/Th2 cell balance toward anti-inflammatory cytokine producing cells.

The invention further provides for the use of any one of a protein ornucleic acid that can reduce the amount, expression or activity ofleptin for the preparation of a medicament for the treatment of animmune-related disorder.

According to one embodiment, an immune-related disorder may be any oneof an inflammatory disorder, an autoimmune-disorder, a graft-rejectionassociated disorder or a fibrotic disorder.

More specifically, an inflammatory disorder may be an intestinalinflammatory disease, preferably, inflammatory bowel diseases (IBD).

In another embodiment, the immune related disorder may be a fibroticdisorder such as hepatic fibrosis, cardiac fibrosis or colon fibrosis.

In yet another embodiment, the immune-related disorder may be anauto-immune disease, for example, arthritis, diabetes or immune-relatedinfertility disorders.

According to another aspect, the invention relates to a method for thetreatment of immune-related disorders in a mammalian subject in need ofsuch treatment, by manipulating NK T cell population of said subject,wherein manipulation of said NK T cell population results in modulationof the Th1/Th2 cell balance, said method comprises the steps of: (a)obtaining NK T cells from said subject; (b) ex vivo educating the NK Tcells obtained in step (a) by culturing the NKT cells in the presence of(i) an antigen or antigens associated with said immune-related immunedisorder; (ii) an antigen presenting cell, preferably DC and (iii)leptin, any homologue, analogue, derivative or functional fragment ofleptin, an expression vector comprising a nucleic acid sequence encodingfor leptin or any functional fragments thereof, a protein, a peptide, ananti-idiotype antibody to leptin, a diphenylethylene compound containingthiazolidinedione or oxazolidinedione, nicotinic acid, a nicotinicester, an antibody specific for leptin, a fragment of an antibody toleptin, an antibody to the leptin receptor, a fragment of an antibody tothe leptin receptor, soluble leptin receptor, a fragment of leptinreceptor, a protein or peptide capable of inhibiting the expression ofleptin, a nucleic acid or an oligonucleotide comprising sequencescomplementary to leptin or leptin receptor mRNA sequences, a leptinanti-sense nucleic aid sequence, a ribozyme, a small interfering RNA(siRNA) specific for leptin, a thiazolidinedione compound, galenin,SOCS-3, a beta 3-adrenoreceptor, or any combination of any of theforegoing.

The invention further provides for a therapeutic composition for thetreatment of an immune-related disorder in a mammalian subject. Thecomposition of the invention comprises as an effective ingredient exvivo educated autologous NK T cells capable of modulating the Th1/Th2cell balance, and optionally further comprising pharmaceuticallyacceptable carrier, diluent, excipient and/or additive.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Leptin induces in-vitro proliferation of NKT cells

Histogram showing proliferation of NKT cells isolated from experimentalgroups A-H. As shown by the figure, leptin induces a significantincrease in in-vitro proliferation of NKT lymphocytes that are incubatedwith HCC-related antigens in the present of DC's.

FIG. 2A-2B Leptin decrease HCC in T cell deficient nude mice

Demonstrative photograph of a mouse that was not administered leptin(FIG. 2A) five weeks after the subcutaneous implantation ofhepatocellular carcinoma showing a large tumor. Mouse that wascontinuously administered leptin (FIG. 2B) developed a significantlysmaller tumor, with a large necrotic center.

FIG. 3 Leptin significantly decrease tumor size in T cell deficient nudemice

Tumors of mouse group A that was administered leptin (top row) weresignificantly smaller in size than tumors of mouse group C that was notadministered leptin (bottom row). The latter group featured a 20%mortality, as compared to 0% in the former group.

FIG. 4A-4B Leptin induces an intense inflammatory response in tumorinterphase areas

Histological evaluation of excised tumors of group C mice (notadministered leptin) revealed a lack of inflammatory response ininterphase areas (FIG. 4A). In comparison, group A mice that wereadministered leptin uniformly developed an intense inflammatory responsein tumor interphase areas (FIG. 4B).

FIG. 5 Leptin induces a significant increase in serum ALT activity 8hours following concanavalin A infusion

Histogram demonstrate the ALT activity of four experimental groups,con+leptin, con, leptin and control.

FIG. 6A-6C leptin induces a significant exacerbation ofconcanavalin-induced hepatitis

Hepatic histological sections showing that leptin induces a significantexacerbation of concanavalin-induced hepatitis (FIG. 6C) in comparisonto concanavalin alone (FIG. 6B). Leptin administration by itself did notproduce any hepatic damage (FIG. 6A).

FIG. 7 Leptin significantly increases the ratio of in-vitro splenocytesecretion of IFN-γ and IL10 (IFN-γ/IL10)

Histogram showing the IFN-γ/IL10 ratio in four different experimentalgroups A-D including con+leptin, con, leptin and control group,respectively.

FIG. 8 Leptin increases inflammation

FIG. 9 rt PCR analysis of peripheral lymphocytes mRNA expression

FIG. 10 Leptin increases natural killer cell cytotoxicity

FIG. 11 Leptin inhibits tumor cell growth

FIG. 12 rtPCR analysis of mRNA expression of STAT2 and SOCS1 in HCCcells

FIG. 13 Leptin decreases the hepatic/splenic NKT cell ratio. Histogramshowing the NKT/lymphocyte ratio in four experimental groups A-Dincluding con+leptin, con, leptin and control group, respectively.

FIG. 14 Leptin significantly decreases tumor weight and volume

FIG. 15 Leptin significantly decreases Hepatocellular Carcinomamortality

FIG. 16 Leptin increases the NK cell population

DETAILED DESCRIPTION OF THE INVENTION

A number of methods of the art of molecular biology are not detailedherein, as they are well known to the person of skill in the art. Suchmethods include site-directed mutagenesis, PCR cloning, expression ofcDNAs, analysis of recombinant proteins or peptides, transformation ofbacterial and yeast cells, transfection of mammalian cells, and thelike. Textbooks describing such methods are e.g., Sambrook et al.,Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory;ISBN: 0879693096, 1989, Current Protocols in Molecular Biology, by F. M.Ausubel, ISBN: 047150338X, John Wiley & Sons, Inc. 1988, and ShortProtocols in Molecular Biology, by F. M. Ausubel et al., (eds.) 3rd ed.John Wiley & Sons; ISBN: 0471137812, 1995. These publications areincorporated herein in their entirety by reference. Furthermore, anumber of immunological techniques are not in each instance describedherein in detail, as they are well known to the person of skill in theart. See e.g., Current Protocols in Immunology, Coligan et al., (eds),John Wiley & Sons. Inc., New York, N.Y.

Leptin is a hormone which has been shown to interact with its cognatereceptor, thereby initiating its cell-signaling pathway. Severaldifferent leptin receptor isoforms are predicted to exist, including along form which has the highest level of expression in regions of thehypothalamus, and specific regions therein, including the arcuatenucleus and the dorso-medial hypothalamus. In vitro and in vivo studiesdemonstrate that leptin activates cytokine-like signal transduction bystimulating the classic JAK-STAT pathway via the long receptor isoform[Ghilardi, N. et al., Proc. Natl. Acad. Sci. USA. 93:6231-6235 (1997);Baumann, H. et al, Proc Natl. Acad. Sci. USA 93:8374-8378 (1996); VaisseC. et al., Nature Genetics 14:95-97 (1996)]. Lack of functional leptinor of long form leptin receptors in the ob/ob and db/db mice,respectively, causes severe obesity [Zhang, et al., Nature 372:425-432(1994); Lee, G. H. et al., Nature 379:632-635 (1996); Chen, H. et al.,Cell 84:491-495 (1996)].

The cloning of the genes encoding leptin [Zhang (1994) ibid.,] and theleptin receptor [Tartaglia, et al., 61183:1263-1271(1995)], and thestudy of these proteins in vivo and in vitro, have dramaticallydemonstrated the importance of this ligand-receptor system in the normalregulation of body weight and energy balance.

In addition to this role which has been proposed to be its primaryfunction, circulating leptin also appears to play an important role inthe neuroendocrine axis [Ahima, R. S., et al., Nature 382:250-252(1996)], including the regulation of reproduction. Administration ofexogenous leptin has been shown to induce the onset of puberty in mice[U.S. patent application Ser. No. 08/749,534, the teachings of which areincorporated herein by reference in its entirety].

The inventors have now shown that leptin is involved inimmuno-modulation of the Th1/Th2 response, and particularly leads toenhancement of the pro-inflammatory response. As shown by Example 1,leptin induces NK T cell proliferation, therefore, leptin could induce apro-inflammatory response via NK T cells.

Thus, in a first aspect, the present invention relates to a method forimmuno-modulation of the Th1/Th2 cell balance, in a subject in needthereof, comprising the step of modulating the levels, the expressionand/or the activity of leptin in said subject.

According to one embodiment, modulation of the Th1/Th2 cell balance bythe method of the invention may be performed by increasing or decreasingthe levels, the activity and/or the expression of leptin in saidsubject.

As shown by the present invention leptin may act as a potentimmuno-modulator by modulating the Th1/Th2 balance in a subjectsuffering from an immune-related disorder. However, it is to beappreciated that the modulation processes regulated by leptin may befurther mediated by different components of the subject's immune system,such as cellular immune reaction elements, humoral immune reactionelements and cytokines. Therefore, leptin may play a dual role in immunemodulation by switching the immune response in the immunogenic ortolerogenic directions. Thus, regulation of the immune response byleptin depends on the environment of such immune response, which isdetermined by different types of stimulations and different signalingpathways. It is noteworthy that leptin is involved in distinctimmunoregulatory mechanisms, and modulates different types of effectorcells and the Th1/Th2 paradigm in immune-mediated disorders.

According to a specific preferred embodiment, modulation of the Th1/Th2cell balance may be mediated by the activation of immuno-regulatorycells selected from the group consisting of NK T cells, antigenpresenting cells and CD4⁺, CD25⁺ T cells, and preferably, NK T cells, byleptin.

The findings of the invention therefore provide new modalities by usingleptin as an immuno-regulator agent.

It is therefore particularly interesting to note that NK T cells, andparticularly, NK1.1 T cells may be involved in keeping a balance betweenanti-inflammatory and pro-inflammatory lymphocytes via cytokinessecretion and/or killing, and may be involved in the determination of Thelper cell differentiation [Arase, H., et al., Eur. J. Immunol. 23:307-310 (1993); Yoshimoto, T, et al., J. Exp. Med. 179:1285-1295 (1994),MacDonald, H. R., et al., J. Exp. Med. 182:633-638 (1995), Seder, R. A.et al., Annu. Rev. Immuno. 12:635-673 (1994), Yoshimoto, T., et al.,Science 270:1845-1847 (1995)]. Multiple signaling pathways wereidentified for NK1.1 T cells activation. It is assumed that NK1.1⁺ Tcells are not stably polarized, and upon different triggers TCRengagement triggers both Th1 and Th2 cytokine secretion from these cells[Bendelac, A., et al., Annu. Rev. Immunol. 15:535-562 (1997); Arase, H.,et al., J. Immunol. 151:546 (1993); Kawamura, T., et al., J. Immunol.160:16-19 (1998), Chen, H., et al., J. Immonol., 159:2240-2249 (1997);Arase, H, et al., Eur. J. Immunol. 23: 307-310 (1998); Yoshimoto, T., J.Exp. Med. 179: 1285-1295 (1994); MacDonald, H. R., J. ibid., (1995)].NK1.1R or IL12R engagement may selectively promote the Th1 secretionparadigm [Bendelac, et al. (1997) ibid.; Arase, H., et al., J. Exp. Med.183:2391-2396 (1996); Hayakawa, T., et al., J. Exp. Med. 176:269-274(1992)].

Therefore, in one specific embodiment, the invention provides for amethod for immuno-modulation resulted by shifting of the Th1/Th2 cellbalance toward the pro-inflammatory cytokine producing cells, in asubject in need thereof. Accordingly, this method comprises the step ofincreasing the levels, the expression and/or the activity of leptin insaid subject.

More particularly, increasing the levels or the expression of leptin ina subject in need thereof may be performed by administering to saidsubject an immuno-modulatory effective amount of a protein or nucleicacid that will increase the amount of leptin, the expression of leptinor leptin activity in the subject. Examples of proteins can comprise butnot be limited to leptin, homologues of leptin, functional derivativesof leptin, leptin fragments and anti-idiotypic antibodies to leptins.Nucleic acids useful for the purpose of increasing the amount,expression or activity of leptin can be designed to express any of theforegoing proteins. Small molecules such as nicotinic acids, nicotinicacid ester or diphenylethylene compounds that contain eitherthiazolidinedione or oxazolidinedione moieties may also find use in thepresent invention. These compounds or reagents may be used alone or theymaybe used in combination with each other.

Homologues of leptin refer to proteins, in which one or more of theamino acid residues of a natural leptin are replaced by different aminoacid residues, or are deleted, or one or more amino acid residues areadded to the natural sequence of leptin, without changing considerablythe activity of the resulting products as compared with the wild typeleptin. These homologues are prepared by known synthesis and/or bysite-directed mutagenesis techniques, or any other known techniquesuitable therefore.

Any such homologue preferably has a sequence of amino acids sufficientlyduplicative of that of leptin, such as to have substantially similaractivity to leptin. One such activity may be the ability of a leptinhomologue to reduce the body weight of ob/ob mice. Thus, it can bedetermined whether any given homologue has substantially the sameactivity as leptin by means of routine experimentation. In aspecifically preferred embodiment, leptin homologue is capable ofmodulating Th1/Th2 cell balance.

In a preferred embodiment, any such protein has at least 40% sequenceidentity or homology with the sequence of either leptin. Morepreferably, it has at least 50%, at least 60%, at least 70%, at least80% or, most preferably, at least 90% sequence identity or homologythereto.

Homologues of leptin polypeptides, which can be used in accordance withthe present invention, or nucleic acid coding therefore, include afinite set of substantially corresponding sequences as substitutionpeptides or polynucleotides which can be routinely obtained by one ofordinary skill in the art, without undue experimentation, based on theteachings and guidance presented herein. For a detailed description ofprotein chemistry and structure, see Schulz, G. E. et al., Principles ofProtein Structure, Springer-Verlag, New York, 1978; and Creighton, T.E., Proteins: Structure and Molecular Properties, W.H. Freeman & Co.,San Francisco. 1983, which are hereby incorporated by reference.

Preferred changes for homologues in accordance with the presentinvention are what are known as “conservative” substitutions.Conservative amino acid substitutions of leptin polypeptides may includesynonymous amino acids within a group which have sufficiently similarphysicochemical properties that substitution between members of thegroup will preserve the biological function of the molecule [Grantham,et al., Science 185:862-864 (1974)]. It is clear that insertions anddeletions of amino acids may also be made in the sequences of leptinwithout altering its function, particularly if the insertions ordeletions only involve a few amino acids, e.g., under thirty, andpreferably under ten, and do not remove or displace amino acids whichare critical to a functional conformation, e.g., cysteine residues[Anfinsen, et al., Science 181:223.230 (1973)]. Proteins produced bysuch deletions and/or insertions come within the purview of the presentinvention.

It should be noted that any homologue of leptin has an amino acidsequence essentially corresponding to that of leptin. The term“essentially corresponding to” is intended to comprehend proteins withminor changes to the sequence of the natural protein which do not affectthe basic characteristics of the natural proteins, particularly insofaras their ability to modulate the immune-system, preferably, towards thepro-inflammatory response. The type of changes which are generallyconsidered to fall within the “essentially corresponding to” languageare those which would result from conventional mutagenesis techniques ofthe DNA encoding these proteins, resulting in a few minor modifications.

“Functional derivatives” as used herein cover derivatives of leptin orits active fragments or fractions and its fusion proteins, which may beprepared from the functional groups which occur as side chains on theresidues or the N- or C-terminal groups, by means known in the art, andare included in the invention as long as they remain pharmaceuticallyacceptable, i.e. they do not destroy the activity of the protein whichis substantially similar to the activity of leptin, and do not confertoxic properties on compositions containing it. These derivatives may,for example, include polyethylene glycol side-chains which may maskantigenic sites and extend the residence of leptin or its activefractions in body fluids. Other derivatives include aliphatic esters ofthe carboxyl groups, amides of the carboxyl groups by reaction withammonia or with primary or secondary amines, N-acyl derivatives of freeamino groups of the amino acid residues formed with acyl moieties (e.g.alkanoyl or carbocyclic aroyl groups) or O-acyl derivatives of freehydroxyl groups (for example that of seryl or threonyl residues) formedwith acyl moieties.

As “functional fragments” of leptin and leptin fusion proteins, thepresent invention covers any fragment or precursors of the polypeptidechain of leptin, or fused proteins containing any such fragment ofleptin, alone or together with associated molecules or residues linkedthereto, e.g., sugar or phosphate residues, or aggregates of any of theabove derivatives, provided said fraction has substantially similaractivity to leptin.

Thus, by “functional fragments” is meant “fragments”, “variants”,“analogs” or “derivatives” of the molecule. A “fragment” of a molecule,such as any of the amino acid sequence of leptin used by the presentinvention is meant to refer to any amino acid subset of the molecule. A“variant” of such molecule is meant to refer to a naturally occurringmolecule substantially similar to either the entire molecule or afragment thereof. An “analog” of a molecule is a homologous moleculefrom the same species or from different species. By “functional” ismeant having same biological function, for example, required forimmuno-modulation.

As indicated above, the terms derivatives and functional derivatives asused herein mean peptides comprising the amino acid sequence of leptin,with any insertions, deletions, substitutions and modifications to thepeptide that do not interfere with the peptides ability to modulateTh1/Th2 response (hereafter referred to as “derivative/s”). A derivativeshould maintain a minimal homology to said amino acid sequence, e.g. notless than 30%. It should be appreciated that the term “insertions” asused herein is meant any addition of amino acid residues to the peptidesused by the invention, between 1 to 50 amino acid residues, preferably,between 20 to 1. amino acid residues and most preferably, between 1 to10 amino acid residues.

As indicated by said preferred embodiment, enhancing the levels ofleptin in a subject in need thereof may be accomplished by the use ofexpression vectors encoding leptin or leptin homologues, provided bygene therapy.

As used herein, the term “nucleic acid” refers to polynucleotides suchas deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA). The terms should also be understood to include, as equivalents,analogs of either RNA or DNA made from nucleotide analogs, and, asapplicable to the embodiment being described, single-stranded anddouble-stranded polynucleotides. “Construct” or “vectors”, as usedherein, encompases vectors such as plasmids, viruses, bacteriophage,integratable DNA fragments, and other vehicles, which enable theintegration of DNA fragments into the genome of the host. Expressionvectors are typically self-replicating DNA or RNA constructs containingthe desired gene or its fragments, and operably linked genetic controlelements that are recognized in a suitable host cell and effectexpression of the desired genes. These control elements are capable ofeffecting expression within a suitable host. Generally, the geneticcontrol elements can include a prokaryotic promoter system or aeukaryotic promoter expression control system. This typically include atranscriptional promoter, an optional operator to control the onset oftranscription, transcription enhancers to elevate the level of RNAexpression, a sequence that encodes a suitable ribosome binding site,RNA splice junctions, sequences that terminate transcription andtranslation and so forth. Expression vectors usually contain an originof replication that allows the vector to replicate independently of thehost cell.

A vector may additionally include appropriate restriction sites,antibiotic resistance or other markers for selection ofvector-containing cells. Plasmids are the most commonly used form ofvector but other forms of vectors which serves an equivalent functionand which are, or become, known in the art are suitable for use herein.See, e.g., Pouwels et al., Cloning Vectors: a Laboratory Manual (1985and supplements), Elsevier, N.Y.; and Rodriguez, et al. (eds.) Vectors:a Survey of Molecular Cloning Vectors and their Uses, Buttersworth,Boston, Mass. (1988), which are incorporated herein by reference.

It should be further appreciated that the invention further encompassesthe use of a host cell transformed or transfected with a constructexpressing leptin or any fragments thereof. Suitable host cells includeprokaryotes, lower eukaryotes, and higher eukaryotes. Prokaryotesinclude gram negative and gram positive organisms, e.g., E. coli and B.subtilis. Lower eukaryotes include yeast, S. cerevisiae and Pichia, andspecies of the genus Dictyostelium. Higher eukaryotes includeestablished tissue culture cell lines from animal cells, both ofnon-mammalian origin, e.g., insect cells and birds, and of mammalianorigin, e.g., human and other primate, and of rodent origin.

“Host cell” as used herein refers to cells which can be recombinantlytransformed with vectors constructed using recombinant DNA techniques. Adrug resistance or other selectable marker is intended in part tofacilitate the selection of the transformants. Additionally, thepresence of a selectable marker, such as drug resistance marker may beof use in keeping contaminating microorganisms from multiplying in theculture medium. Such a pure culture of the transformed host cell wouldbe obtained by culturing the cells under conditions which require theinduced phenotype for survival.

As used herein, the term “transfection” means the introduction of anucleic acid, e.g., an expression vector, into a recipient cells bynucleic acid-mediated gene transfer. “Transformation”, as used herein,refers to a process in which a cell's genotype is changed as a result ofthe cellular uptake of exogenous DNA or RNA.

“Cells”, “host cells” or “recombinant cells” are terms usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cells but to the progeny or potential progenyof such a cell. Because certain modification may occur in succeedinggeneration due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

According to a specifically preferred embodiment, the method of theinvention is particularly useful for shifting of the Th1/Th2 cellbalance toward the proinflammatory cytokine producing cells a mammaliansubject suffering of an immune-related disorder.

More preferably, shifting of the Th1/Th2 cell balance toward theproinflammatory cytokine producing cells may be particularlyadvantageous for immune-related disorder such as malignant proliferativedisorder a disorder caused by immuno-suppression and an infection causedby a pathogenic agent.

According to one embodiment, a malignant proliferative disorder may beany one of solid and non-solid tumor selected from the group consistingof carcinoma, sarcoma, melanoma, leukemia and lymphoma. In canceroussituations, modulation of the NK T cells by leptin, as showed by theExamples may be in the direction of inducing a pro-inflammatoryresponse. As used herein to describe the present invention, “cancer”,“tumor” and “malignancy” all relate equivalently to a hyperplasia of atissue or organ. If the tissue is a part of the lymphatic or immunesystems, malignant cells may include non-solid tumors of circulatingcells. Malignancies of other tissues or organs may produce solid tumors.In general, the methods and compositions of the present invention may beused in the treatment of non-solid and solid tumors, for example,carcinoma, sarcoma, melanoma, leukemia, and lymphoma.

Malignancies that may find utility in the present invention can comprisebut are not limited to hematological malignancies (including leukemia,lymphoma and myeloproliferative disorders), hypoplastic and aplasticanemia (both virally induced and idiopathic), myelodysplastic syndromes,all types of paraneoplastic syndromes (both immune mediated andidiopathic) and solid tumors.

More specifically, according to a preferred embodiment, modulation ofleptin or leptin activity, could be used for the treatment or inhibitionof non-solid cancers, e.g. hematopoietic malignancies such as all typesof leukemia, e.g. acute lymphocytic leukemia (ALL), acute myelogenousleukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenousleukemia (CML), myelodysplastic syndrome (MDS), mast cell leukemia,hairy cell leukemia, Hodgkin's disease, non-Hodgkin's lymphomas,Burkitt's lymphoma and multiple myeloma, as well as for the treatment orinhibition of solid tumors such as tumors in lip and oral cavity,pharynx, larynx, paranasal sinuses, major salivary glands, thyroidgland, esophagus, stomach, small intestine, colon, colorectum, analcanal, liver, gallbladder, extraliepatic bile ducts, ampulla of vater,exocrine pancreas, lung, pleural mesothelioma, bone, soft tissuesarcoma, carcinoma and malignant melanoma of the skin, breast, vulva,vagina, cervix uteri, corpus uteri, ovary, fallopian tube, gestationaltrophoblastic tumors, penis, prostate, testis, kidney, renal pelvis,ureter, urinary bladder, urethra, carcinoma of the eyelid, carcinoma ofthe conjunctiva, malignant melanoma of the conjunctiva, malignantmelanoma of the uvea, retinoblastoma, carcinoma of the lacrimal gland,sarcoma of the orbit, brain, spinal cord, vascular system,hemangiosarcoma and Kaposi's sarcoma.

According to another particular embodiment, as also shown by theExamples, the method of the invention may be advantageous forHepatocellular carcinoma (HCC). Hepatocellular carcinoma (HCC) is theseventh leading cause of cancer-related mortality in men and the ninthin women. [Bosch, F. X. and Munoz, N. In: Etiology, patology andtreatment of hepatocellular carcinoma in America. Advaces in appliedtechnology series. Tabor E, Dibiscegile A M, Purcell Rh (Eds.), Gulf,Houston (1991)]. The prevalence of the disease is steadily increasing inthe western world, mainly due to a growing pool of longstanding chronicHCV carriers. An estimated one million deaths are caused annually fromHCC [El Serag, H. B. et al., Ann. Intern. Med. 18:139(10):817-23(2003)]. HCC is resistant to systemic chemotherapy and radiationtreatment. Chemoembolisation, ethanol injection, radiofrequencyablasion, and tumor resection offer a slight prognostic advantage andare mainly used as palliative measures. Orthotopic liver transplantationmay offer cure in small tumors, but is rarely employed [Llovet, J. M. etal., Lancet. 6:362(9399): 1907-17 (2003)]. Therefore, modulation of theimmune response towards a pro-inflammatory response by the use ofleptin, is advantageous.

In another preferred embodiment, the subject in need of modulating theTh1/Th2 cell balance toward the pro-inflammatory cytokine producingcells by the method of the invention may suffer from anyimmuno-suppressive disorder, for example immune suppression caused byinfection of immunodeficiency virus, preferably, HIV, or alternatively,immunosuppression caused by chemotherapy. Chemotherapeutic treatment inmalignancy is often associated with life-threatening opportunisticinfections, resulting from potent inhibition of various arms of theimmune system. Often, the intensity and duration of chemotherapeutictreatment are limited by these side effects. Stimulation of the immunesystem, optionally by the coadministration of leptin may enabletreatment with higher doses and more potent combinations ofchemotherapeutic agents, thereby reducing the risks ofmedication-induced immune suppression.

According to another embodiment, shifting of the Th1/Th2 cell balancetoward the pro-inflammatory cytokine producing cells by the method ofthe invention may be advantageous in a subject suffering of aninfection, for example infection caused by a pathogenic agent.

Reference to pathogenic agents includes a prokaryotic microorganism, alower eukaryotic microorganism, a complex eukaryotic organism, a virus,fungi, prions, parasite, yeast and venoms.

A prokaryotic microorganism includes bacteria such as Gram positive,Gram negative and Gram variable bacteria and intracellular bacteria.Examples of bacteria contemplated herein include the speices of thegenera Treponema sp., .Borrelia sp., Neisseria .sp., Legionella sp.,Bordetella sp., Escherichia sp., Salmonella sp., Shigella sp.,Klebsiella sp., Yersinia sp., Vibrio sp., Hemophilus sp., Rickettsiasp., Chlamydia sp., Mycoplasma sp., Staphylococcus sp., Streptococcussp., Bacillus sp., Clostridium sp., Corynebacterium sp.,Proprionibacterium sp., Mycobacterium sp., Ureaplasma sp. and Listeriasp.

Particularly preferred species include Treponema pallidum, Borreliaburgdorferi, Neisseria gonorrhea, Neisseria meningitidis, Legionellapneumophila, Bordetella pertussis, Escherichia coli, Salmonella typhi,Salmonella typhimurium, Shigella dysenteriae, Klebsiella pneumoniae,Yersinia pestis, Vibrio cholera, Hemophilus influenzae, Rickettsiarickettsii, Chlamydia trachomatis, Mycoplasma pneumoniae, Staphylococcusaureus, Streptococcus pneumoniae, Streptococcus pyogenes, Bacillusanthracis, Clostridium botulinum, Clostridium tetani, Clostridiumperfringens, Corynebacterium diphtheriae, Proprionibacterium acnes,Mycobacterium tuberculosis, Mycobacterium leprae and Listeriamonocytogenes.

A lower eukaryotic organism includes a yeast or fungus such as but notlimited to Pneumocystis carinii, Candida albicans, Aspergillus,Histoplasma capsulatum, Blastomyces dermatitidis, Cryptococcusneoformans, Trichophyton and Microsporum.

A complex eukaryotic organism includes worms, insects, arachnids,nematodes, aemobe, Entamoeba histolytica, Giardia lam blia, Trichomonasvaginalis, Trypanosoma brucei gambiens, Trypanosoma cruzi, Balantidiumcoli, Toxoplasma gondii, Cryptosporidium or Leishmania.

The term “viruses” is used in its broadest sense to include viruses ofthe families adenoviruses, papovaviruses, herpesviruses: simplex,varicella-zoster, Epstein-Barr, CMV, pox viruses: smallpox, vaccinia,hepatitis B, rhinoviruses, hepatitis A, poliovirus, rubellavirus,hepatitis C, arboviruses, rabiesvirus, influenzaviruses A and B,measlesvirus, mumpsvirus, HIV, HTLV I and II.

The term “fungi” includes for example, fungi that cause diseases such asringworm, histoplasmosis, blastomycosis, aspergillosis, cryptococcosis,sporotrichosis, coccidioidomycosis, paracoccidio-idoinycosis, andcandidiasis.

The term parasite includes, but not limited to, infections caused bysomatic tapeworms, blood flukes, tissue roundworms, ameba, andPlasmodium, Trypanosoma, Leishniania, and Toxoplasma species.

The invention further provides for a method for the treatment of animmune-related disorder in a subject in need thereof, comprising thestep of administering to said subject an immuno-modulatory effectiveamount of a protein or nucleic acid that will increase the amount ofleptin, the expression of leptin or leptin activity in the subject.Examples of proteins can comprise but not be limited to leptin,homologues of leptin, functional derivatives of leptin, leptin fragmentsand anti-idiotypic antibodies to leptins. Nucleic acids useful for thepurpose of increasing the amount, expression or activity of leptin canbe designed to express any of the foregoing proteins. Small moleculessuch as nicotinic acids, nicotinic acid ester or diphenylethylenecompounds that contain either thiazolidinedione or oxazolidinedionemoieties may also find use in the present invention. These compounds orreagents may be used alone or they maybe used in combination with eachother.

As used herein in the specification and in the claims section below, theterm “treat” or treating and their derivatives includes substantiallyinhibiting, slowing or reversing the progression of a condition,substantially ameliorating clinical symptoms of a condition orsubstantially preventing the appearance of clinical symptoms of acondition.

As used herein, “an immuno-modulatory amount” or “an amount sufficientto modulate the Th1/Th2 balance” means an amount necessary to achieve aselected therapeutic result. For example, in this particular embodimentan effective amount of the composition of the invention will modulatethe Th1/Th2 balance toward pro-inflammatory cytokine producing cells.

Thus, in one embodiment, the effective amount administered to a subjectsuffering from an immune-related disorder may be an amount sufficientfor shifting the Th1/Th2 cell balance toward pro-inflammatory cytokineproducing cells.

According to another embodiment, the method of the invention isparticularly applicable for the treatment of an immune-related disordersuch as a malignant proliferative disorder, a disorder caused byimmuno-suppression or an infection caused by a pathogenic agent asdescribed above.

More particularly, the method of the invention is particularlyapplicable for the treatment of a malignant proliferative disorder suchas solid and non-solid tumors selected from the group consisting ofcarcinoma, sarcoma, melanoma, leukemia and lymphoma.

According to a specifically preferred embodiment, the method of theinvention is for the treatment of Hepatocellular carcinoma (HCC).

According to another embodiment, the method of the invention is suitablefor the treatment of immuno-suppression caused by infection of animmunodeficiency virus, preferably, HIV, or caused by chemotherapy.

Still further, the method of the invention is intended for the treatmentof an infection caused by a pathogenic agent, for example, bacterialpathogens, viruses, fungi, parasites or yeast.

In another aspect, the invention relates to the use of a protein ornucleic acid that will increase the amount, expression or activity ofleptin for the preparation of a composition for shifting the Th1/Th2cell balance toward pro-inflammatory cytokine producing cells.

The invention further relates to the use a protein or nucleic acid thatwill increase the amoun, expression or activity of leptin for thepreparation of a medicament for the treatment of an immune-relateddisorder.

According to one embodiment of said aspect, the immune-related disordermay be a malignant proliferative disorder, a disorder caused byimmuno-suppression or an infection caused by a pathogenic agent.

Specifically, a malignant proliferative disorder may be solid andnon-solid tumors selected from the group consisting of carcinoma,preferably, Hepatocellular carcinoma (HCC), sarcoma, melanoma, leukemiaand lymphoma.

In another embodiment, the immune related disorder may beimmuno-suppression, for example, caused by infection of immunodeficiencyvirus, preferably, HIV, or alternatively, by chemotherapy.

In yet another embodiment, the immune related disorder may be infectioncaused by a pathogenic agent selected from the group consisting ofbacterial pathogens, viruses, fungi, parasites and yeast.

The role of leptin in modulation of the Th1/Th2 response and in theinduction of a pro-inflammatory response, enables the manipulation ofthis element such that, for example, its neutralization will favorinduction of an anti-inflammatory response.

Therefore, in a third aspect, the invention relates to a method forimmuno-modulation of the immune system resulted by shifting the Th1/Th2cell balance toward the anti-inflammatory cytokine producing cells, in asubject in need thereof. Such method comprises the step of decreasingthe amounts, the expression and/or the activity of leptin in saidsubject.

According to one embodiment, decreasing the amounts, the expression orthe activity of leptin in said subject by the method of the inventionmay be performed by administering to a subject in need thereof, animmuno-modulatory effective amount of proteins or nucleic acids wherethe proteins can comprise but not be limited to leptin fragments,antibodies to leptin, fragments of antibodies to leptin, soluble leptinreceptor, fragments of the leptin receptor, antibodies to the leptinreceptor and fragments of antibodies to leptin receptor, galanin and theSOCS-3 gene product and the nucleic acids can comprise but not belimited to oligonucleotides or nucleic acids that act as antisense,ribozymes or siRNA or that code for expression of antisense RNA,ribozymes or siRNA. These compounds or reagents may be used alone orthey maybe used in combination with each other.

The term “antibody” is meant to encompass polyclonal antibodies,monoclonal antibodies (mAbs), chimeric antibodies (e.g., humanizedantibodies) and antibody fragments that retain the biological activityof specific binding to leptin, such as Fab, Fab′, F(ab′)2 and Fv. Alsoencompassed are single-chain antibodies (sFvs). These antibody fragmentslack the Fc portion of an intact antibody, clear more rapidly from thecirculation and can have less non-specific tissue binding than an intactantibody. These fragments are produced by well-known methods in the art,for example by proteolytic cleavage with enzymes such as papain (toproduce Fab fragments) or pepsin (to produce F(ab′)2 fragments).

Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of animals immunized with an antigen. Amonoclonal antibody (mAb) contains a substantially homogenous populationof antibodies specific to antigens, which population containssubstantially similar epitope binding sites. MAbs may be obtained bymethods known to those skilled in the art. See, for example Kohler andMilstein, Nature 256:495-497, 1975; U.S. Pat. No. 4,376,110; Ausubel etal, eds., Current Protocols in Molecular Biology, Green PublishingAssoc. and Wiley Interscience, N.Y., 1987, 1992; and Harlow and LaneAntibodies. A Laboratory Manual Cold Spring Harbor Laboratory, 1988;Colligan et al, eds., Current Protocols in Immunology, Greene PublishingAssoc. and Wiley Interscience, N.Y., 1992, 1993; the contents of whichreferences are incorporated entirely herein by reference. Suchantibodies can be of any immunoglobulin class including IgG, IgM, IgE,IgA, and any subclass thereof A hybridoma producing a mAb of the presentinvention can be cultivated in vitro, in situ, or in vivo. Production ofhigh titers of mAbs in vivo or in situ makes this the presentlypreferred method of production.

Chimeric antibodies which include humanized antibodies, are moleculeswherein different portions of which are derived from different animalspecies, such as those having variable regions derived from a murine mAband a human immunoglobulin constant region. Chimeric antibodies areprimarily used to reduce immunogenicity in application and/or toincrease yields in production, for example. Chimeric antibodies andmethods for their production are known in the art [Cabilly, et al.,Proc. Natl. Acad. Sci. USA 81:3273-3277 (1984); Morrison, et al., Proc.Natl. Acad. Sci. USA 81:6851-6855 (1984); Boulianne, et al., Nature312:643-646 (1984)]. These references are entirely incorporated hereinby reference.

Typically, antibodies that may be used for reducing leptin levels orfunction by the methods of the present invention are high affinityanti-leptin antibodies, and fragments or regions thereof that havepotent inhibiting and/or neutralizing activity in vivo against leptin.Such antibodies can include those generated by immunization usingpurified recombinant leptin or peptide fragments thereof.

Methods for determining antibody specificity and affinity can be foundin Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1988; Colligan et al., eds.,Current Protocols in Immunology, Greene Publishing Assoc. and WileyInterscience, NY., 1992, 1993; and Muller, Meth. Enzymol., 92:589-6011983; which references are entirely incorporated herein by reference.

The generation of polyclonal antibodies against proteins is described inChapter 2 of Current Protocols in Immunology, Wiley and Sons Inc.Monoclonal antibodies may be prepared from B cells taken from the spleenor lymph nodes of immunized animals, in particular rats or mice, byfusion with immortalized B cells under conditions which favor the growthof hybrid cells. For fusion of murine B cells, the cell line Ag-8 ispreferred. The technique of generating monoclonal antibodies isdescribed in many articles and textbooks, such as the above-notedChapter 2 of Current Protocols in Immunology. Spleen or lymph node cellsof these animals may be used in the same way as spleen or lymph nodecells of protein-immunized animals, for the generation of monoclonalantibodies as described in Chapter 2 therein. The techniques used ingenerating monoclonal antibodies are further described by Kohler andMilstein, Nature 256:495-497, (1975), and in U.S. Pat. No. 4,376,110.

As indicated above, for future clinical applications, where the antibodyis a monoclonal antibody, it may be improved, through a humanizationprocess, to overcome the human antibody to mouse antibody response.Rapid new strategies have been developed recently for antibodyhumanization which may be applied for such an antibody. Thesetechnologies maintain the affinity, while retaining antigen and epitopespecificity of the original antibody Rader, C., et al., Proc. Natl.Acad. Sci. 95:8910-8915 (1998); Mateo, C., et al., Immunotechnology3:71-81 (1997)]. A “humanized” antibody, in which, for example animal(say murine) variable regions are fused to human constant regions, or inwhich murine complementarity-determining regions are grafted onto ahuman antibody. Unlike, for example, animal derived antibodies,“humanized” antibodies often do not undergo an undesirable reaction withthe immune system of the subject.

Thus, as used herein, the term “humanized” and its derivatives refers toan antibody which includes any percent above zero and up to 100% ofhuman antibody material, in an amount and composition sufficient torender such an antibody less likely to be immunogenic when administeredto a human being. It is understood that the term “humanized” reads alsoon human derived antibodies or on antibodies derived from non humancells genetically engineered to include functional parts of the humanimmune system coding genes, which therefore produce antibodies which arefully human.

Reducing the amount, expression and activity of leptin by the method ofthe invention may be also achieved by the use of particular nucleic acidsequences, such as anti-sense sequence, siRNA and a catalytic nucleicacid sequence such as ribozyme that are specific for leptin or theleptin receptor. The term “siRNAs” refers to short interfering RNAs. Theterm “RNA interference” or “RNAi” refers to the silencing or decreasingof gene expression by siRNAs. It is the process of sequence-specific,post-transcriptional gene silencing in animals and plants, initiated bysiRNA that is homologous in its duplex region to the sequence of thesilenced gene. The gene may be endogenous or exogenous to the organism,present integrated into a chromosome or present in a transfection vectorthat is not integrated into the genome. The expression of the gene iseither completely or partially inhibited. RNAi may also be considered toinhibit the function of a target RNA; the function of the target RNA maybe complete or partial. RNA interference (RNAi) is a mechanism involvingdouble-stranded RNA (dsRNA) molecules and resulting inposttranscriptional sequence-specific silencing of gene expression.

As indicated above, ribozymes may also be used. Ribozymes are RNAmolecules having an enzymatic activity which is able to repeatedlycleave other separate RNA molecules in a nucleotide base sequencespecific manner. Several basic varieties of naturally-occurringenzymatic RNAs are known presently. Each can catalyze the hydrolysis ofRNA phosphodiester bonds in-trans (and thus can cleave other RNAmolecules) under physiological conditions. In general, enzymatic nucleicacids act by first binding to a target RNA. Such binding occurs throughthe target binding region of a enzymatic nucleic acid which is held inclose proximity to an enzymatic region or catalytic region of themolecule that acts to cleave the target RNA. Thus, the enzymatic nucleicacid first recognizes and then binds a target RNA through complementarybase-pairing, and once bound to the correct site, acts enzymatically tocut the target RNA. Strategic cleavage of such a target RNA will destroyits ability to direct synthesis of an encoded protein, and according tothe present invention, the expression of leptin. After an enzymaticnucleic acid has bound and cleaved its RNA target, it is released fromthat RNA to search for another target and can repeatedly bind and cleavenew targets. Thus, a single ribozyme molecule is able to cleave manymolecules of target RNA. In addition, the ribozyme is a highly specificinhibitor of gene expression, with the specificity of inhibitiondepending not only on the base-pairing mechanism of binding to thetarget RNA, but also on the mechanism of target RNA cleavage. Singlemismatches, or base-substitutions, near the site of cleavage cancompletely eliminate catalytic activity of a ribozyme.

According to a specific embodiment, a subject may be a mammalian subjectsuffering of an immune-related disorder.

More particularly, the immune-related disorder may be any one of aninflammatory disorder, an autoimmune-disorder, a graft-rejectionassociated disorder or a fibrotic disorder.

Therefore, in a further embodiment, proteins and nucleic acids thatdecrease the amount, expression or activity of leptin may be used fortreatment of or amelioration of inflammatory symptoms in any disease,condition or disorder where immune and/or inflammation suppression isbeneficial such as, but not limited to, treatment of or amelioration ofinflammatory symptoms in the joints, musculoskeletal and connectivetissue disorders, or of inflammatory symptoms associated withhypersensitivity, allergic reactions, asthma, atherosclerosis, otitisand other otorhinolaryngological diseases, dermatitis and other skindiseases, posterior and anterior uveitis, conjunctivitis, opticneuritis, scleritis and other immune and/or inflammatory ophthalmicdiseases.

Diseases characterized by airway inflammation affect a substantialproportion of the population. These diseases include asthma and chronicobstructive pulmonary disease (COPD). In the European Union, COPD andasthma, together with pneumonia, are the third most common cause ofdeath. The production of cytokines and growth factors in response toirritants, infectious agents and inflammatory mediators play animportant role in the initiation, perpetuation and inhibition of acuteand chronic airway inflammation.

Airway inflammation is associated with excessive production and activityof several mediators and cytokines released by inflammatory and residentcells in the airways. Now it is clear that the epithelium is not only animportant target for the action of mediators of inflammation, but alsoan active participant in the inflammatory process itself. Bronchialepithelial cells are able to recruit inflammatory cells to the airwaysthrough the release of chemoattractants, to direct inflammatory cellmigration across the epithelium through the expression of cell adhesionmolecules, and to regulate the inflammatory activity of other cellsthrough the release of mediators, like cytokines, chemokines,arachidonic acid metabolities and relaxant and contractile factors.

In one embodiment, an inflammatory disorder may be an intestinalinflammatory disease, preferably, an inflammatory bowel diseases (IBD).

Inflammatory bowel diseases (IBD) are common gastrointestinal disorders,that can be perceived as being the result of a dysbalance betweenTh1-pro-inflammatory, and Th2-anti-inflammatory subtypes of immuneresponses [Strober, W., et al., Immunol Today 18:61-64 (1997); Neurath,M., et al., J. Exp. Med. 183:2605-2616 (1996)].

There are several extra-intestinal manifestations that accompany IBD,for example: autoimmune phenomena since immune complexes have a role intarget organ damage, immunosuppressive agents such as glucocorticoids,azathioprine, methotrexate and cyclosporin are used to alleviate thedisease [Podolsky, D. K., et al., New Engl. J. Med., 325:928-935(1991);Strober, W., et al., In Clinical Immunology, Mosby, St. Louis. R. R.Rich, Editor, 1401-14281-2 (1995)]. Patients with IBD have antibodiesagainst components of colon cells and several different bacterialantigens. These antigens gain access to the immune system as aconsequence of epithelial damage [Hibi, S., et al., Clin. Exp. Immunol.54:163-168 (1983); Das, K. M., et al., Gastroenterology 98:464-69(1990)]. Abnormalities of T cell-mediated immunity, including coetaneousanergy and diminished responsiveness to T cell stimuli, have also beendescribed in these patients [Chiba, M., et al., Gut, 22:177-182 (1981);Raedler, A., et al., Clin. Exp. Immunol. 60:518-526 (1985)]. Inaddition, changes in mucosal cell mediated immunity were identified,including increased concentrations of mucosal IgG cells and changes in Tcells subsets, suggesting antigen stimulation [Dasgupta, A., et al., Gut35:1712-17 (1994); Takahashi, F., et al., J. Clin. Invest. 76:311-318(1985)]. Exposure of target antigens after infectious, immune, or toxicdamage, leads to activation of mucosal immune cells resulting incytokines that lead to mucosal inflammatory response [Neurath, M., etal., J. Exp. Med., 183:2605-2616 (1996)]. Secretion of pro-inflammatorycytokines such as IFNγ, contributes to an increase in mucosalpermeability, and has been described in animal models of IBD [Strober,W., et al., Immunol. Today 18:61-64. (1997)]. Similarly, an increase incollagen synthesis mediated by IL1 and IL6 can be detected in theseanimals [Strober, W., et al., ibid.]. A Th1-mediated granulomatouscolitis model has been established by the adoptive transfer of normalCD45RB T cells from Balb/C mice into CB-17 scid mice. CD4 cells fromCD45RB were shown to prevent the disease when injected together with theCD45RB population. This prevention could be reversed by addingantibodies to TGFJB1 [Sadlack, B., et al., Cell 75:253-261 (1993);Powrie, F., et al., Immunity 1:553-562 (1994)].

Furthermore, the present invention discloses the use of leptinfragments, antibodies to leptin, fragments of antibodies to leptin,soluble leptin receptor, fragments of the leptin receptor, antibodies tothe leptin receptor and fragments of antibodies to leptin receptor,galanin and the SOCS-3 gene product as well as oligonucleotides ornucleic acids that act as antisense, ribozymes or siRNA or that code forexpression of antisense RNA, ribozymes or siRNA or any combination ofthe foregoing when an immune-mediated disorder comprises an allergy,asthma or a parasitic infection. The present invention may find furtheruse when said immune-mediated disorder comprises an over-reactive immuneresponse directed against infectious agents such as a virus or abacterium. Often in these diseases, production of autoreactiveantibodies and/or autoreactive T lymphocytes can be a result of thisover-response. For example, when IgE is overproduced or when a diseaseis Th2 dependent, the reaction itself can be detrimental to the organismas, for example, seen with some parasitical diseases or withmycobacterial infections such as TBC or leprosy. An autoimmune responsemay also occur as a manifestation of a viral or bacterial infection andmay result in severe tissue damage, as for example, lymphocyticchoriomeningitis virus (LCMV) infections or the destructive hepatitiscaused by Hepatitis B infection.

In another preferred embodiment, decreasing the levels or activity ofleptin by the methods of the invention are useful for treatment of oramelioration of an autoimmune disease such as, but not limited to,Eaton-Lambert syndrome, Goodpasture's syndrome, Greave's disease,Guillain-Barr syndrome, autoimmune hemolytic anemia (AIHA), hepatitis,insulin-dependent diabetes mellitus (IDDM), systemic lupus erythematosus(SLE), multiple sclerosis (MS), myasthenia gravis, plexus disorders e.g.acute brachial neuritis, polyglandular deficiency syndrome, primarybiliary cirrhosis, rheumatoid arthritis, scleroderma, thrombocytopenia,thyroiditis e.g. Hashimoto's disease, Sjbgren's syndrome, allergicpurpura, psoriasis, mixed connective tissue disease, polymyositis,dermatomyositis, vasculitis, polyarteritis nodosa, polymyalgiarheumatica, Wegener's granulomatosis, Reiter's syndrome, Behget'ssyndrome, ankylosing spondylitis, pemphigus, bullous pemphigoid,dennatitis herpetiformis, insulin dependent diabetes, inflammatory boweldisease, ulcerative colitis and Crohn's disease.

The present invention further provides for a method for the treatment ofan immune-related disorder in a subject in need thereof. The method ofthe invention comprises the step of administering to said subject animmuno-modulatory effective amount of proteins or nucleic acids wherethe proteins can comprise but not be limited to leptin fragments,antibodies to leptin, fragments of antibodies to leptin, soluble leptinreceptor, fragments of the leptin receptor, antibodies to the leptinreceptor and fragments of antibodies to leptin receptor, galanin and theSOCS-3 gene product and the nucleic acids can comprise but not belimited to oligonucleotides or nucleic acids that act as antisense,ribozymes or siRNA or that code for expression of antisense RNA,ribozymes or siRNA. These compounds or reagents may be used alone orthey maybe used in combination with each other.

It should be noted that composition dosages administered by the methodsof the invention may be in any amount that sufficient to modulate theTh1/Th2 balance. It is understood by the skilled artisan that thepreferred dosage would be individualized to the patient following goodlaboratory practices and standard medical practices. As used herein, “anamount sufficient to modulate the Th1/Th2 balance” means an amountnecessary to achieve a selected result. For example, in the foregoingembodiment an effective amount of the composition of the invention willmodulate the Th1/Th2 balance toward anti-inflammatory cytokine producingcells.

Thus, according to one embodiment, an effective amount may be an amountsufficient for shifting the Th1/Th2 cell balance towardanti-inflammatory cytokine producing cells.

In another embodiment, the method of the invention is intended for thetreatment of an immune-related disorder such as inflammatory disorder,an autoimmune-disorder a graft-rejection associated disorder or afibrotic disorder.

According to a specific embodiment, the method of the invention isparticularly advantageous for the treatment of an inflammatory disorder,preferably, an intestinal inflammatory disease and most preferably,inflammatory bowel diseases (IBD).

According to another specific embodiment, the method of the invention issuitable for the treatment of a fibrotic disorder such as any one ofhepatic fibrosis, cardiac fibrosis or colon fibrosis.

Still further, the method of the invention may be applicable for thetreatment of auto-immune disease, such as arthritis, diabetes orimmune-related infertility disorders.

According to another aspect, the invention relates to the administrationproteins or nucleic acids that will decrease the amount of leptin, theexpression of leptin or leptin activity for the preparation of acomposition that shifts the Th1/Th2 balance towards anti-inflammatorycytokine producing cells. Examples of proteins can comprise but not belimited to leptin fragments, antibodies to leptin, fragments ofantibodies to leptin, soluble leptin receptor, fragments of the leptinreceptor, antibodies to the leptin receptor and fragments of antibodiesto leptin receptor, galanin and the SOCS-3 gene product. Examples ofnucleic acids can comprise but not be limited to oligonucleotides ornucleic acids that act as antisense, ribozymes or siRNA or that code forexpression of antisense RNA, ribozymes or siRNA. These compounds orreagents may be used alone or they maybe used in combination with eachother.

The invention further provides for the use administration of proteins ornucleic acids that will decrease the amount of leptin, the expression ofleptin or leptin activity for the preparation of a medicament for thetreatment of an immune-relate disorder. Examples of proteins cancomprise but not be limited to leptin fragments, antibodies to leptin,fragments of antibodies to leptin, soluble leptin receptor, fragments ofthe leptin receptor, antibodies to the leptin receptor and fragments ofantibodies to leptin receptor, galanin and the SOCS-3 gene product.Examples of nucleic acids can comprise but not be limited tooligonucleotides or nucleic acids that act as antisense, ribozymes orsiRNA or that code for expression of antisense RNA, ribozymes or siRNA.These compounds or reagents may be used alone or they maybe used incombination with each other.

According to one embodiment, an immune-related disorder may be aninflammatory disorder, an autoimmune-disorder, a graft-rejectionassociated disorder or a fibrotic disorder.

More specifically, an inflammatory disorder may be an intestinalinflammatory disease, preferably, inflammatory bowel diseases (IBD).

In another embodiment, the immune related disorder may be a fibroticdisorder such as hepatic fibrosis, cardiac fibrosis or colon fibrosis.

In yet another embodiment, the immune-related disorder may be anauto-immune disease, for example, arthritis, diabetes and immune-relatedinfertility disorders.

In yet a further aspect, the invention relates to a method for thetreatment of immune-related disorders in a mammalian subject in need ofsuch treatment, by manipulating NK T cell population of said subject,wherein manipulation of said NK T cell population results in modulationof the Th1/Th2 cell balance, said method comprises the steps of: (a)obtaining NK T cells from said subject; (b) ex vivo educating the NK Tcells obtained in step (a) by culturing the NKT cells in the presence of(i) an antigen or antigens associated with said immune-related immunedisorder; (ii) an antigen presenting cell, preferably DC and (iii)leptin, a leptin homologue, a modified leptin, a functional leptinfragment, a nucleic acid construct expressing any of the foregoing orany combination thereof such that the resulting educated NK T cells havethe capability of modulating the Th1/Th2 cell balance; and (c)re-introducing to said subject the educated NK T cells obtained in step(b) which are capable of modulating the Th1/Th2 cell balance.

The NK T cells educated by the method of the invention can be obtainedfrom bone marrow, liver, spleen, or uterus, but can also be obtainedfrom the peripheral blood, by cytopheresis, a procedure by which a largenumber of white cells are obtained, while other blood components arebeing simultaneously transferred back to the subject.

It should be noted that several cell types appear to be capable ofserving as APC's (antigen presenting cells), including dendritic cells(DC), activated B cells, and activated macrophages. In accordance withthe invention, the APCs used for co-culturing with NK T cells, arepreferably autologous cells and in some illustrative preferredembodiments the antigen-presenting cell may be a dendritic cell (DC). Itis understood that one of skill in the art will recognize that otherantigen presenting cells may be useful in the invention, such as B cellsactivated by lipopolysaccharide, whole spleen cells, peripheral bloodmacrophages, fibroblasts or non-fractionated peripheral bloodmononuclear cells (PBMC). Therefore, the invention is not limited to theexemplary cell types which are specifically mentioned and exemplifiedherein.

Co-culturing of the NK T cells in the presence of peripheral lymphocytesfrom patients suffering from the same immune-related disorder or from atreated subject, is also contemplated in the present invention. In orderto obtain lymphocytes from a subject, particularly a human subject,blood may be drawn from the patient by the cytopheresis methodsdescribed above.

As indicated above, the NK T cells are co-cultured with APC's in thepresence of leptin, and an antigen or antigens associated with theimmune related disorder in which the subject suffers from.

It should be appreciated that the antigen or antigens associated with animmune-related disorder can be native or non-native with regards to thesubject. They can be natural or synthetic, modified or unmodified, wholeor fragments thereof. Fragments can be derived from synthesis asfragments or by digestion or other means of modification to createfragments from larger entities. Such antigen or antigens comprise butare not limited to proteins, glycoproteins, enzymes, antibodies,histocompatibility determinants, ligands, receptors, hormones,cytokines, cell membranes, cell components, viruses, viral components,viral vectors, non-viral vectors, whole cells, tissues or organs. Theantigen can consist of single molecules or mixtures of diverseindividual molecules. The antigen can present itself within the contextof viral surface, cellular surface, membrane, matrix, or complex orconjugated with a receptor, ligand, antibody or any other bindingpartner. Such antigen or antigens can be introduced to the subject aloneor with agent or agents that could further contribute to uptake,stability, reactivity or targeting.

Polymerization and degradation, fractionation and chemical modificationare all means that can be used to alter the properties of a particularantigen in terms of potential immune responses. These small segments,fragments or epitopes can either be isolated or synthesized.

As a non-limiting example, such antigen may be a combination ofdifferent antigens derived from body extracts, preferably of the subjectto be treated or from a subject suffering from the same disorder.

The invention further provides for a therapeutic composition for thetreatment of an immune-related disorder in a mammalian subject. Thecomposition of the invention comprises as an effective ingredient exvivo educated autologous NK T cells capable of modulating the Th1/Th2cell balance, and optionally further comprising pharmaceuticallyacceptable carrier, diluent, excipient and/or additive.

According to one embodiment, the educated autologous NK T cell used forthe composition of the invention may be obtained by ex vivo culture byculturing the NKT cells in the presence of (i) an antigen or antigensassociated with said immune-related immune disorder; (ii) an antigenpresenting cell, preferably DC and (iii) leptin, a leptin homologue, amodified leptin, a functional leptin fragment, a nucleic acid constructexpressing any of the foregoing or any combination thereof.

Methods for in vitro storage, growth or expansion of cells prior toeducation or to transfer of the cells to the treated subject, are wellknown to practitioners of the art. When the educated NK T cells intendedfor use in a transfer are derived from a donor, these cells may alsoundergo storage, growth or expansion in vivo or in vitro as describedabove.

It is to be appreciated that the NK T cells may be educated in vivo aswell, via any of the methods described above, they can be modulatedprior to or at any point of time following exposure to the APC, leptinand antigens related to said disorder.

Cell therapy may be by injection, e.g., intravenously, or by any of themeans described herein above. Neither the time nor the mode ofadministration is a limitation on the present invention. Cell therapyregimens may be readily adjusted taking into account such factors as thepossible cytotoxicity of the educated cells, the stage of the diseaseand the condition of the patient, among other considerations known tothose of skill in the art.

It should be noted that any of the components used by the differentmethods of the invention, e.g., proteins or nucleic acids that increasethe amount, expression or activity of leptin, proteins or nucleic acidsthat decrease the amount, expression or activity of leptin, host cellscontaining such proteins or nucleic acids or educated NKT cells, may byadministered in a single dose, or alternatively in multiple doses. Thesecomponents may be administered by a single route of administration oralternatively, by at least two different routes of administration.

The proteins or nucleic acids that increase the amount, expression oractivity of leptin, proteins or nucleic acids that decrease the amount,expression or activity of leptin, host cells containing such proteins ornucleic acids or educated NKT cells, may be administered directly to thesubject to be treated or, depending on the size of the compound, it maybe desirable to conjugate them to a carrier prior to theiradministration. Therapeutic formulations may be administered in anyconventional dosage formulation. Formulations typically comprise atleast one active ingredient, as defined above, together with one or moreacceptable carriers thereof.

Each carrier should be both pharmaceutically and physiologicallyacceptable in the sense of being compatible with the other ingredientsand not injurious to the patient. Formulations include those suitablefor oral, rectal, nasal, or parenteral (including subcutaneous,intramuscular, intravenous and intradermal) administration. Theformulations may conveniently be presented in unit dosage form and maybe prepared by any methods well known in the art of pharmacy. Thenature, availability and sources, and the administration of all suchcompounds including the effective amounts necessary to produce desirableeffects in a subject are well known in the art and need not be furtherdescribed herein.

More specifically, the said proteins or nucleic acids that increase theamount, expression or activity of leptin, proteins or nucleic acids thatdecrease the amount, expression or activity of leptin, host cellscontaining such proteins or nucleic acids or educated NKT cells, may beadministered by a route selected from oral, intravenous, parenteral,transdermal, subcutaneous, intravaginal, intranasal, mucosal,sublingual, topical and rectal administration and any combinationsthereof.

Although the method of the invention is particularly intended for thetreatment of immune-related disorders in humans, other mammals areincluded. By way of non-limiting examples, mammalian subjects includemonkeys, equines, cattle, canines, felines, mice, rats and pigs.

The proteins or nucleic acids that increase the amount, expression oractivity of leptin, proteins or nucleic acids that decrease the amount,expression or activity of leptin, host cells containing such proteins ornucleic acids or educated NKT cells, may be administered in a singledose, or alternatively in multiple doses. These components may beadministered by a single route of administration or alternatively, by atleast two different routes of administration. More specifically, saidcomponents may be administered by a route selected from oral,intravenous, parenteral, transdermal, subcutaneous, intravaginal,intranasal, mucosal, sublingual, topical and rectal administration andany combinations thereof.

For treating a mammalian subject suffering of an immune-relateddisorder, for example, cancer, the leptin, homologues of leptin,functional derivatives of leptin, leptin fragments, anti-idiotypicantibodies to leptins, nucleic acid constructs that express any of theforegoing proteins, host cells containing such constructs or educatedNKT cells disclosed in various embodiments of the present invention canbe administered in a variety of ways. By way of a non-limiting example,these components, may be delivered intravenously, or into a body cavityadjacent to the location of a solid tumor, such as the intraperitonealcavity, or injected directly into or adjacent to a solid tumor.

It should be noted that all components and compositions used by themethods of the invention may optionally include sustained-releasematrices, such as biodegradable polymers, to form therapeuticcompositions. A sustained-release matrix, as used herein, is a matrixmade of materials, usually polymers, which are degradable by enzymaticor acid/base hydrolysis or by dissolution. Once inserted into the body,the matrix is acted upon by enzymes and body fluids. Thesustained-release matrix desirably is chosen from biocompatiblematerials such as liposomes, Polylactides (polylactic acid),polyglycolide (polymer of glycolic acid), polylactide co-glycolide(co-polymers of lactic acid and glycolic acid) polyanhydrides,poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitinsulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides,nucleic acids, polyamino acids, amino acids such as phenylalanine,tyrosine, isoleucine, polynucleotides and polyvinylpyrrolidone.

The immuno-modulating pharmaceutical compositions according to thepresent invention may be a solid, liquid or aerosol and may beadministered by any known route of administration. Examples of solidtherapeutic compositions include pills, creams, and implantable dosageunits. The pills may be administered orally or the therapeutic creamsmay be administered topically.

The implantable dosage units may be administered locally, for example ata tumor site, or may be implanted for systemic release of thetherapeutic composition, for example subcutaneously. Examples of liquidcompositions include compositions adapted for injection subcutaneously,intravenously, intraarterially, and compositions for topical andintraocular administration. Examples of aerosol compositions includeinhaler composition for administration to the lungs.

The leptin, homologues of leptin, functional derivatives of leptin,leptin fragments, anti-idiotypic antibodies to leptins, nucleic acidconstructs that express any of the foregoing proteins, host cellscontaining such constructs or educated NKT cells disclosed in variousembodiments of the present invention can be administered by standardroutes. In general, the combinations may be administered by the topical(including buccal and sublingual), or parenteral (includingsubcutaneous, intraperitoneal, intramuscular, intravenous, intradermal,intracerebral, intracerebroventricular, intracranial, intraspinal,intratracheal, and epidural), transdermal, intravaginal, intrauterine,oral, rectal, ophthalmic (including intravitreal or intracameral), orintranasal administration. Osmotic mini-pumps may also be used toprovide controlled delivery of high concentrations of leptin, or leptinderivatives, to the site of interest, such as directly into a metastaticgrowth.

The magnitude of therapeutic dose of the various compositions used inthe present invention will of course vary with the group of patients(age, sex, etc.), the nature of the condition to be treated and with theroute administration and as such, dosage will be determined by theattending physician.

The pharmaceutical forms suitable for injection use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringeability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi.

The prevention of the action of microorganisms can be brought about byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminum monostearate andgelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above.

In the case of sterile powders for the preparation of the sterileinjectable solutions, the preferred method of preparation arevacuum-drying and freeze drying techniques which yield a powder of theactive ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

The pharmaceutical compositions of the invention generally comprise abuffering agent, an agent which adjusts the osmolarity thereof, andoptionally, one or more pharmaceutically acceptable carriers, excipientsand/or additives as known in the art. Supplementary active ingredientscan also be incorporated into the compositions. The carrier can besolvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), suitable mixtures thereof and vegetable oils. Theproper fluidity can be maintained, for example, by the use of a coating,such as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants.

Local administration to the area in need of treatment is also includedand may be achieved by, for example, local infusion during surgery,topical application, direct injection into the inflamed joint, directlyonto the eye, etc.

For oral administration, the pharmaceutical preparation may be in liquidform, for example, solutions, syrups or suspensions, or in solid form astablets, capsules and the like. For administration by inhalation, thecompositions are conveniently delivered in the form of drops or aerosolsprays. For administration by injection, as indicated above, theformulations may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers with an added preservative.

The compositions of the invention can also be delivered in a vesicle,for example, in liposomes. In another embodiment, the compositions canbe delivered in a controlled release system.

The amount of the therapeutic or pharmaceutical composition of theinvention which is effective in the treatment of a particular disease,condition or disorder will depend on the nature of the disease,condition or disorder and can be determined by standard clinicaltechniques. In addition, in vitro assays as well in vivo experiments mayoptionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the seriousness of the disease, conditionor disorder, and should be decided according to the judgment of thepractitioner and each patients circumstances. Effective doses may beextrapolated from dose-response curves derived from in vitro or animalmodel test systems.

Disclosed and described, it is to be understood that this invention isnot limited to the particular examples, methods steps, and compositionsdisclosed herein as such methods steps and compositions may varysomewhat. It is also to be understood that the terminology used hereinis used for the purpose of describing particular embodiments only andnot intended to be limiting since the scope of the present inventionwill be limited only by the appended claims and equivalents thereof.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, an and “the” include plural referentsunless the content clearly dictates otherwise.

Throughout this specification and the Examples and claims which follow,unless the context requires otherwise, the word “comprise”, andvariations such as “comprises” and “comprising”, will be understood toimply the inclusion of a stated integer or step or group of integers orsteps but not the exclusion of any other integer or step or group ofintegers or steps.

The following examples are representative of techniques employed by theinventors in carrying out aspects of the present invention. It should beappreciated that while these techniques are exemplary of preferredembodiments for the practice of the invention, those of skill in theart, in light of the present disclosure, will recognize that numerousmodifications can be made without departing from the spirit and intendedscope of the invention.

Materials & Methods

NKT Lymphocyte and Dendritic Cell Isolation

Donor mice were sacrificed at day 1 of the experiment and spleniclymphocytes were isolated and red blood cells removed as previouslydescribed [Trop, S. et al., Hepatology 29(3):746-55 (1999)]. Spleenswere crushed through a stainless mesh (size 60, Sigma Chemical Co., St.Louis Mo.). Cell suspension was placed in a 50 ml tube for 3 minutes andwashed twice in cold PBS (1,250 rpm for 10 minutes), and debris removed.Cells were re-suspended in PBS, and placed through a nylon meshpresoaked in PBS, and unbound cells were collected. Cells were washedtwice in 45 ml PBS (1,250 rpm at room temperature). The viability bytrypan blue staining was expected to be more than 95%. Cell separationwas performed using Magnetic Cell Sorting (MACS). NKT cells wereisolated using both anti CD3 and anti-NK1.1 beads, while dendritic cellswere isolated using anti CD11a beads, in accordance with themanufacturer's instructions (Miltenyl Biotec, Bergisch Gladbach,Germany).

Flow Cytometry Analysis for Determination of Natural Killer CellPopulation

Following natural killer cell isolation, triplicates of 2-5×10⁵cells/500 μl PBS were put into Falcon 2052 tubes incubated with 4 ml of1% BAS for 10 minutes, and centrifuged at 1400 rpm for 5 minutes. Cellswere resuspended in 10 μl FCS with 1:20 CY5-conjugated CD45 antibody andPE-conjugated anti-pan NK antibody (eBioscience, USA), and mixed every10 minutes for 30 minutes. Cells were washed twice in 1% BSA, and keptin 4° C. unitl reading. For the control group, only 5 μl of 1% BSA wasadded. Analytical cell sorting was performed on 1×10⁴ cells from eachgroup with a fluorescence-activated cell sorter (FACSTAR plus, BectonDickinson). Only live cells were counted, and background fluorescencefrom non-antibody-treated lymphocytes was deducted from the levelsobtained. Gates were set on forward- and side-scatters to exclude deadcells and red blood cells. The data was analyzed with Consort 30two-color contour plot program (Becton Dickinson, Oxnard, Calif.), usingthe CELLQuest program.

HCC Cell Culture

Mouse hepatoma cell line HEPA 1-6 (CRL 1830), was grown in culture asmonolayers in a medium supplemented with nonessential aminoacids and 10%heat inactivated fetal bovine serum.

The human Hepa 3B hepatocellular carcinoma cell line was obtained fromAmerican type culture collection (ATCC), VA. Cell were grown asmonolayers in cultures containing DMEM medium, supplemented with 10%heat-inactivated fetal bovine serum, 1% L-glutamine, nonessential aminoacids, penicillin and streptomycin.

Preparation of HCC Lysate

0.4×10⁶ HEPA 1-6 or HEP-3B cells were separated, placed in Eppendorftubes, and centrifuged at 250 g for 5 minutes. The pellet was separatedand subjected to three cycles of freeze-thaw in liquid nitrogen. Proteinquantification was performed using commercial kits, followingmanufacturer's instructions.

NKT Proliferation Using Radioactive Thymidine Incorporation

Isolated NKT lymphocytes and dendritic cells were cultured in microwellplates (Sterilin Co.), with or without HCC related antigens and elevateddoses of leptin, in a total volume of 0.2 ml RPMI 1640 culture medium,supplemented with 100 mg/ml penicillin, 100 mg/ml streptomycin, 2 mML-glutamine, with 5×10⁻⁵ M 2ME, and 10% FCS. After 24 h in a 37° C.humidified 5% CO₂ incubator, 1 mmCi of ³H TdR (5 Ci/nmol, NuclearResearch Center, Negev, Israel) was added to each well. Cells werecollected 16 h later on paper filters, using a multiple sample harvester(Titertek Cell Harvester 530; Flow Laboratories, McClean, Va.).Radioactivity was measured by a liquid scintillation counter. Backgroundresults were subtracted.

Cytokine Measurement

The content of each plate was centrifuged and the supernatant fluid wascollected. Supernatant cytokine levels (and serum cytokine levels,including leptin, IL10, IL12, IL4, TNF-x, and TGF-β) were measured by a“sandwich” ELISA method, using Genzyme Diagnostic kits (GenzymeDiagnostics, Mass., USA) according to the manufacturer's instructions.

STAT and SMAD Western Blot Analysis

Expression of the transcription factors STAT (signal transducer andactivator of transcription) 1, 3, 4 and 6, p-STAT 1, 3, 4, 6, and SMADand p-SMAD 1-4 in splenocytes was determined by Western blot analysis ofprotein extraction of splenocytes from each plate. Splenocytes werelysed in 100 μl of lysis solution (Sigma). Proteins (100 μg/lane) wereresolved by electrophoresis on SDS-polyacrylamide (7.5%) gels, andelectroblotted to nitrocellulose membranes (Schleicher & Scuell,Germany). Probing with a polyclonal rabbit anti-mouse antibody for thedifferent tested STAT and SMAD proteins (Santa Cruz Biotechnology) wasfollowed by addition of horseradish peroxidaseconjugated goatanti-rabbit IgG (Jackson Immuno Research, PA, USA).

Determination of the Presence of Leptin Receptors on NKT Cells

NKT cells were cultured on six well plates, and were washed and lysed inlysis buffer. Spleens were crushed through a stainless mesh (size 60,Sigma Chemical Co., St Louis Mo.). Cell suspension was placed in a 50 mltube for 3 minutes and washed twice in cold PBS (1,250 rpm for 10minutes), and debris was removed. Cells were re-suspended in PBS, andplaced through a nylon mesh presoaked in PBS, and unbound cells werecollected. Cells were washed twice in 45 ml PBS (1,250 rpm at roomtemperature). For splenocyte isolation, 20 ml of histopaque 1077 (SigmaDiagnostics, St. Louis, Mo.) was slowly placed underneath the cellssuspended in 7 ml of PBS, in a 50-ml tube. The tube was centrifuged at1,640 rpm for 15 minutes at room temperature. Cells at the interfacewere collected, diluted in a 50-ml tube, and washed twice with ice-coldPBS (1,250 rpm for 10 minutes). The viability by trypan blue stainingwas more than 95%. Cell separation was performed using Magnetic CellSorting (MACS). For NKT cells, both anti CD3 and anti-NK1.1 were used(Miltenyl Biotec, Bergisch Gladbach, Germany).

Cell lysate was transferred to micro centrifuge tubes and clarified bycentrifugation. The supernatant was collected and the protein contentwas determined by a BCA protein assay kit for Western blotting analysis,equal amounts of protein were separated by sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis [8%] and were transferred onto anitrocellulose membrane. The membrane was immuno-blotted with goatanti-leptin receptor antibodies (Santa Cruz, USA).

Mice Groups

8-week old male nude mice, SCID mice, and SCID-beige mice were purchasedfrom Jackson Laboratories (Bar Harbor, Me.). All animals were housed inlaminar flow hoods in sterilized cages, given irradiated food andsterile acidified water, and kept on regular 12 hour light-dark cycles.Food and water were administered ad libitum. Mice were weighed and foodintake was recorded every two days. Mice were sacrificed on day 30 ofthe experiment by cervical dislocation, under isoflurane anesthesia. Allanimal experiments were carried out in accordance with the guidelines ofthe Hebrew University-Hadassah Instituional Committee for care and useof laboratory animals and with the committee's approval. Mice thatshowed signs of distress, lost 10% of body weight between measurements,or more than 25% of their initial body weight, were sacrificed.

Induction of HCC

1×10⁸ HCC cells (of HEPA 1-6 origin) were injected intraperitoneallyinto wildtype c57bl and leptin-deficient ob/ob mice in order todetermine the effect of leptin on HCC growth in these mice. Monitoringof tumor growth was performed weekly by mouse weighing and determinationof tumor diameter by calipers. Any mouse that featured a loss of over20% in body weight, exhibited signs of distress, or had an estimatedtumor size of more than 15% of animal weight, was sacrificed.

Leptin Administration

Recombinant mouse leptin was purchased from R&D Co, and dissolvedaccording to manufacturer's instructions. Leptin was administered in twodaily intraperitoneal injections, at a dose of 0.5 μg/gram body weightper injection, as illustrated in Table 3 (see Example 2).

Pathological Examination

Hematoxylin/eosin staining of paraffin-embedded liver and tumor sectionswas performed. Sections were examined by two experienced pathologists,blinded to the criteria of the experiment.

Splenic and Hepatic Lymphocyte Isolation

Splenocytes were isolated and red blood cells removed as previouslydescribed [Shibolet, O. et al., J Leukoc Biol. 75(1):76-86 (2003)].Intrahepatic lymphocytes were isolated from all groups of mice at theend of the study. The inferior vena cava was cut above the diaphragm andthe liver was flushed with 5 ml of cold PBS until it became pale. Theconnective tissue and gall bladder were removed, and livers were placedin a 10-ml dish in cold sterile PBS. Livers and spleens were crushedthrough a stainless mesh (size 60, Sigma Chemical Co., St. Louis Mo.).Cell suspension was placed in a 50-ml tube for 3 minutes and washedtwice in cold PBS (1,250×rpm for 10 minutes), and debris was removed.Cells were re-suspended in PBS, cell suspension was placed through anylon mesh presoaked in PBS, and unbound cells were collected. Cellswere washed twice in 45 ml PBS (1,250×rpm in room temperature). Forliver and spleen lymphocyte isolation 20 ml of histopague 1077 (SigmaDiagnostics, St. Louis, Mo.) was slowly placed underneath the cellssuspended in 7 ml of PBS, in a 50-ml tube. The tube was centrifuge at1,640 rpm for 15 minutes at room temperature. Cells at the interfacewere collected, diluted in a 50-ml tube, and washed twice with ice-coldPBS (1,250 rpm for 10 minutes). Approximately 1×106 cells/mouse liverwere recovered. The viability by trypan blue staining was expected to bemore than 95%.

Flow Cytometry Analysis for Determination of Lymphocyte Subpopulations

Immediately following lymphocyte isolation, triplicates of 2-5×10⁴cells/500 μl PBS were put into Falcon 2052 tubes incubated with 4 ml of1% BSA for 10 minutes, and centrifuged at 1400 rpm for 5 minutes. Cellswere resuspended in 10 μl FCS with 1:20 FITC-anti mouse NK1.1 antibody(NKR-P1C, Pharmingen, USA), and mixed every 10 minutes for 30 minutes.Cells were washed twice in 1% BSA, and kept at 4° C. until reading. Forthe control group, only 5 μl of 1% BSA were added. Analytical cellsorting was performed on 1×10⁴ cells from each group with afluorescence-activated cell sorter (FACSTAR plus, Becton Dickinson).Only live cells were counted, and background fluorescence fromnon-antibody-treated lymphocytes was deducted from the levels obtained.Gates were set on forward- and side-scatters to exclude dead cells andred blood cells. The data was analyzed with Consort 30 two-color contourplot program (Becton Dickinson, Oxnard, Calif.), or the CELLQuestprogram.

Lymphocyte RNA Isolation and Semi-quantitative rtPCR

Total RNA from 10×10⁶ splenocytes of each mouse of each experimentalgroup was isolated and transcribed into complementary DNA using PromegaReverse Transcriptase Kit (USA). PCR products were obtained after 35cycles of amplification with an annealing temperature of 56-62 andvisualized by ethidium bromide staining after agarose electrophoresis.RtPCR products were semiquantified by visual analysis afternormalization against the Actin internal control. Primer sequences usedfor mouse target gene detection are depicted in table 2.

Natural Killer Cell Cytotoxicity Determination

10³ Natural killer cells were incubated for 4 hours with severalconcentrations of target YAC cells (NK:YAC ratio of 1:5 to 1:0.04) inthe presence of 0, 0.01, 0.1, 1 mcg/ml mouse leptin. Cytotoxicity wasdetermined by measuring lactate dehydrogenase (LDH) activity released inthe media following 4 hours of incubation, using the CytoTox96nonradioactive assay (Promega) and quantitated by measuring wavelengthabsorbance at 490 nm. Data were normalized to a maximal release of LDHupon lysis of YAC cells with (100%) and are corrected for spontaneousbaseline LDH release from YAC cells, NK cells and the buffer.

Natural Killer Cell Proliferation Assay

Splenocytes were collected and prepared as described above. Naturalkiller cells were produced from splenocytes using double passage throughmagnetic beads conjugated to monoclonal hamster antimouse pan-NKantibody (Miltenyi Biotec, Bergisch Gladbach, Germany), in accordancewith the manufacturer's instructions. Natural killer cells were seededin RPMI medium containing 10% fetal calf serum at a density of 1×104cells/16-mm well of a 24-well plate, in quadruplicates, in the presenceof 500 ng/ml recombinant mouse IL-2. Each quadruplicate was added with0, 0.01, 0.1, or 1 mcg/ml mouse highly purified leptin. After 5 days ofincubation, methyl-H³ thymidine was added to all wells (1 μCi/ml,Amersham Pharmacia Biotech, Little Chalfont, UK). Cell cultures wereharvested following 16 hours. Data were given as mean stimulationindices of triplicates, expressed as counts per minute (CPM).

HCC Cell Proliferation

HEPA 3B Cells were cultured in DMEM medium containing 10% fetal calfserum at a density of 1×103 cells/16-mm well of 24-well plate, inquadruplicate, in the presence of 0, 0.01, 0.1, and 1 mcg/ml humanhighly purified leptin. After 5 days of incubation, methyl-H³ thymidinewas added to all wells (1 μCi/ml, Amersham Pharmacia Biotech, LittleChalfont, UK). HCC cell cultures were harvested following 16 hours. Datawere given as mean stimulation indices of triplicates, expressed ascounts per minute (CPM).

HCC Cell & Natural Killer Cell Proliferation Assay

Mouse natural killer cells wer isolated from splenocytes as describedabove. Natural killer cells were irradiated with 3000 rads prior toincubation to avoid proliferation. A combination of 1×10³ HEPA 3B Cellsand 1×104 mouse derived natural killer cells were cultured in RPMImedium containing 10% fetal calf serum and 500 ng/ml IL2 at a density of1×103 cells/16-mm well of a 24-well plate, in quadruplicate, in thepresence of 0, 0.01, 0.1, and 1 mcg/ml human highly purified leptin.After 5 days of incubation, methyl-H³ thymidine was added to all wells(1 μCi/ml, Amersham Pharmacia Biotech, Little Chalfont, UK). HCC cellcultures were harvested following 16 hours. Data were given as meanstimulation indices of triplicates, expressed as counts per minute(CPM).

HCC Cell mRNA Determination

HEPA 3B Cells were cultured in quadruplicates for 1, 2, and 6 hours inRPMI medium containing 10% fetal calf serum at a density of 1×106cells/16-mm well of a 24-well plate, in the presence of 0.5 mcg/ml, 5mcg/ml, 50 mcg/ml human highly purified leptin. Total RNA was isolatedand transcribed into complementary DNA using Promega ReverseTranscriptase Kit (USA). PCR products were obtained after 35 cycles ofamplification with an annealing temperature of 56-62 and visualized byethidium bromide staining after agarose electrophoresis. RtPCR productswere semiquantified by visual analysis after normalization against theActin internal control. Primer sequences used for mouse target genedetection are depicted in table 3.

In vitro Lymphocyte Incubation

1×10⁵ splenic lymphocytes from each mouse were cultured in 96-wellplates in the presence of concanavalin A (used here as a T lymphocyteactivator), for 24 hours. Subsequently, plates were centrifuged and thesupernatant fluid removed. Determination of supernatant levels of IFN-g,IL10, 1L12 was performed using ‘sandwich ELISA’ (described above).

HCC in T Cell Deficient Mice

The above experiments were repeated in T cell deficient nude mice. 1×10⁷HEP-3B HCC cells were injected subcutaneously, with or without theaddition of leptin, as outlined in Table 1 below. Follow-up parameterswere identical to the ones described above.

TABLE 1 In vivo nude mice experimental groups (n = 10) Group ATen-week-old male nude mice + HCC + 1 μg/g/day leptin Group BTen-week-old male nude mice + HCC Group C Ten-week-old male nude mice +1 μg/g/day leptin Group D Ten-week-old male C57BL/6 mice

EXAMPLES Example 1

In-vitro Addition of Increasing Doses of Leptin Results in EnhancedAPC-dependent NKT Lymphocyte Proliferation

In order to assert the immunomodulatory effects of leptin on regulatorycell (NKT and dendritic cell) proliferation and cytokine secretionprofile, the inventors have performed a series of in-vitro experimentsassessing the effect of leptin on NKT lymphocyte proliferation,particularly in the presence of HCC related antigens. NKT cells wereharvested from wildtype c57bl mice using double beading techniques(anti-CD3+anti-NK1.1 & anti-CD11a, for NKT and dendritic cells,respectively), as described in Experimental Procedures. Triplicates of1×10⁵ NKT cells were either incubated alone, or with the addition of1×10⁴ dendritic cells (DC). A crude lisate of HEPA 1-6 HCC cells (madeout from 5×10⁶ cells), and increasing doses of leptin were added to eachplate, as indicated in Table 2, which lists all experimental groups. Thesame experiment was later repeated using a human HEP-3B HCC lysate, inorder to determine whether the same response occurs in the presence ofhuman HCC antigens.

Plates were incubated for one day, and NKT cell proliferation wasdetermined using thymidine uptake techniques. As shown by FIG. 1, higherdoses of leptin resulted in greater NKT cell proliferation, inparticularly in plates where NKT cells were incubated together with DC's(270 CPM, P<0.05). These results indicate that incubation of NKT cellswith escalating doses of leptin, in the presence of tumor relatedantigens, results in increased NKT proliferation, when these cells areincubated together with dendritic cells. This suggests that leptinfeatures a direct, dose-dependent, pro-proliferative influence on NKTcells, through activation of DC's.

TABLE 2 Experimental groups (triplicates) Group Cells Leptin HCCantigens Group A NKT cell No leptin 5 × 10⁶ HEP 1-6 cell lysate Group BNKT cell + DC No leptin 5 × 10⁶ HEP 1-6 cell lysate Group C NKT cell 0.5ng/ml 5 × 10⁶ HEP 1-6 cell lysate leptin

Example 2

Leptin Leads to Induction of HCC in T Cell Deficient Nude Mice whichResults in a Substantial Increment in Tumor Growth

In vitro and in vivo experiments were next performed in order todetermine the presence of leptin receptors on HCC cells grown in cellculture. Since it was previously found that enhancement of tumor cellgrowth by leptin occurs through activation of the JAK/STAT pathway, theinventors further examined the effect of leptin addition on expressionof STAT and STAT intracellular proteins. It should be noted that invitro, leptin receptor mRNA expression and leptin-induced expression ofsignal transducer and activators (STAT) 1-6, suppressor of cytokinesignaling (SOCS) 1-4, and cytokine-inducible S—H protein (C15) weredetermined in human HEPA-3B HCC cells. Subsequently, HCC was induced inleptin-deficient ob/ob mice [Schwartz, J. et al., Clin. Invest.98(5):1101-6 (1996)] and their lean littermates, with or without theaddition of leptin, as illustrated in Table 3, and the effect of leptinand leptin deficiency on HCC growth was determined. To analyze thedirect and immune-mediated effects of leptin, HCC was induced as controlin T cell deficient nude mice, with or without the addition of exogenousleptin.

As shown by FIG. 2, Induction of HEP 3B human HCC in immune deficientnude mice resulted in a significant decrease in tumor growth (FIG. 2B)in the presence of leptin (although these tumors showed a large necroticcenter), in comparison to mice that were not treated with leptin (FIG.2A). It should be noted that changes were notable after only five weeksof follow-up.

TABLE 3 In vivo ob/ob experimental Eroups (n = 10) Group A Ten-week-oldmale ob/ob mice + PBS Group B Ten-week-old male C57BL/6 mice + PBS GroupC Ten-week-old male ob/ob mice + 1 μg/g/day leptin Group D Ten-week-oldmale C57BL/6 mice + 1 μg/g/day leptin Group E Ten-week-old male ob/obmice + HCC + PBS Group F Ten-week-old male C57BL/6 mice + HCC + PBSGroup G Ten-week-old male ob/ob mice + HCC + 1 μg/g/day leptin Group HTen-week-old male C57BL/6 mice + HCC + 1 μg/g/day leptin

A comparative experiment, in which tumors of mice treated with leptinand mice which were not treated with leptin, is demonstrated by FIG. 3.As shown by the figure, tumors of mice that were treated with leptin(top row) were significantly smaller then tumors taken from mice whichwere not treated with leptin (bottom row).

However, histological evaluation of excised tumors of both groups (FIG.3), clearly demonstrated that mice treated with leptin uniformlydeveloped an intense inflammatory response in tumor interphase areas(FIG. 3B), whereas tumors of mice which were not treated with leptin,showed no inflammatory response.

These results clearly demonstrate the feasibility of the use of leptinfor decreasing tumor size, and indicate that reduction of tumor size byleptin may be a result of an immuno-modulatory effect of leptin oninduction of a pro-inflammatory response.

Example 3

Leptin Induces a Potent Pro-inflammatory Immune Response in theConcanavalin A Hepatic Damage Model

To assess the immunomodulatory role of leptin on NKT cell dependenthepatic disease, the effect of leptin was assessed in the concanavalinA-induced hepatitis model [Watanabe, Y. et al., Hepatology 24(3):702-10(1996)]. Wildtype c57bl/6 mice were divided into 4 groups, as shown inTable 4. Group A and C mice were administered with two dailyintraperitoneal injections of 0.5 mg/g body weight leptin for one week.On day eight, mice of groups A and B were intravenously administeredwith 20 mg/g concanavalin A (conA). Eight hours later all mice weresacrificed.

Leptin administration resulted in significantly exacerbatedconcanavalin-induced hepatitis. Group A mice featured significantlyelevated serum ALT activity (4578±2226 u/l, FIG. 5) in comparison togroup B mice (2035±446 u/l, P<0.05). Hepatic histology from group A micedemonstrated significantly increased hepatic necrosis and inflammationas compared to group B mice (FIG. 6). The administration of leptinwithout the induction of concanavalin A hepatitis did not in itselfresult in elevated ALT (alanine amino transferase) levels (114±101 u/l)or hepatic necrosis (FIGS. 5 and 6, respectively).

TABLE 4 Preliminary experiment B groups (N + 10) Group LeptinConcanavalin A Group A + + Group B − − Group C + + Group D − −

In vitro exposure to concanavalin A of cultured splenocytes from allmice groups at the end of the experiment resulted in a significantincrease in the ratio of supernatant levels of IFN-γ/IL10 in group Amice that were treated with both leptin and Concanavalin A (0.6±0.5) asshown by FIG. 5, in comparison to mice of group B (0.17±0.1), C(0.32±0.08), and D (0.15±0.07, P=0.07). Hepatic NKT lymphocytes weresignificantly lower among group A mice (4±1.2%), in comparison to groupB (10±0%), C (12±8%) & D (30±8.5%), possibly reflecting theactivation-induced apoptosis.

These results suggest that leptin functions as a pro-inflammatory immunemodulating agent, inducing exacerbation of Th1 immune response,pro-inflammatory cytokine secretion, possibly through NKT lymphocyteactivation.

Example 4

In-vivo Effect of Leptin Administration on Hepatocellular Carcinoma inAthymic Mice

Leptin administration resulted in a significant inhibition ofhepatocellular carcinoma growth and improved survival in athymic mice.Differences in tumor size between leptin-administered andsaline-administered mice were noted after two weeks of tumorimplantation. After six weeks of follow-up, mean tumor volume inleptin-administered mice (1.04±0.12 cm³) was significantly lower than insaline-administered mice (1.9±0.9 cm³, P<0.001, FIG. 3). Mean tumorweight was significantly lower in leptin-administered mice (0.31±0.05grams) as compared to saline administered mice (0.63±0.37gr, P=0.002).Mortality was significantly lower among leptin-administered than amongsaline-administered mice (0% vs. 20% mortality on week 6, respectively,P<0.0001).

Macroscopically, tumors of leptin-administered mice featured a smallsolid mass and a large necrotic center, whereas tumors insaline-administered mice were composed of a much larger solid mass and asignificantly smaller area of necrosis (FIG. 9). Microscopically, tumorsin leptin-administered mice featured a dense inflammatory infiltrate,mainly in the interphase area. The inflammatory exudates was composed ofa mixture of lymphocytes and neutrophilles (FIG. 8). Such intra-tumorinfiltrate was not notable in saline-administered mice.Leptin-administered and saline-administered mice in control groups B & Ddid not feature tumor growth or mortality.

Example 5

In-vivo Effect of Leptin Administration on Natural Killer Subsets

Peripheral natural-killer cell population was expanded inleptin-administered, HCC-implanted mice (6.16±2.19%) in comparison tosaline administered mice (3.25±0.67%, P=0.03). A similar increase in NKcell population was noted in leptin-administered control group B ascompared to saline-administered group D (1.27±2.73 vs. 0.58±0.37%, ingroups B and D, respectively, P=0.007, FIG. 4). No statisticallysignificant differences in the hepatic/splenic natural killer cell ratiowere noted between leptin and saline administered mice (1.02±1.41,0.45±0.64, 1.05±1.05, 0.25±0.24, for groups A-D respectively, P=NS).

Example 6

In-vivo Effect of Leptin Administration on Natural Killer Cell mRNAExpression

Semiquantitative rtPCR analyis of peripheral lymphocyte mRNA expressionof several pro and anti-inflammatory factors revealed significantlydecreased expression of CIS protein in leptin-administered as comparedto saline administered mice (FIG. 9). Lymphocyte mRNA expression ofSTAT1-6, SOCS 1-4 did not differ between groups.

Example 7

In vivo Effect of Leptin Administration on Cytokine Profile

Serum leptin levels were significantly higher in leptin treated group Aand B mice (7.05 ng/ml and 6.29 ng/ml, respectively) than saline treatedgroup C and D mice (3.24 ng/ml and 1.57 ng/ml, respectively, P<0.01). Nosignificant difference between groups was noted in any of the otherserum cytokine levels (P=NS).

Example 8

In-vivo Effect of Leptin Administration on Hepatocellular Carcinoma inSCID and in SCID-Beige Mice

To further determine the role of natural killer cells inleptin-associated tumor suppression, the in-vivo experiments wererepeated using T&B cell deficient SCID mice and T, B & natural killercell deficient SCID-Beige mice. Leptin administration to SCID miceresulted in similar tumor inhibition to that observed in Athymic nudemice. Numbers. In natural killer cell-deficient SCID beige mice, on theother hand, tumor volume and tumor weight were significantly larger(5.05±2.65 cm³ & 2.73±1.61 grams in SCID-Beige saline administered micecompared to 1.9±0.9 cm³ & 0.63±0.37 grams in saline-administered nudemice, p<0.001 for both parameters). Leptin and saline-administered SCIDBeige mice suffered of 30% & 40% mortality, respectively, throughout theexperimental period. In contrast to nude and SCID mice,leptin-administration to SCID-beige mice resulted in no tumor inhibitoryeffect (mean tumor volume of 4.62±2.29 cm³ & weight of 2.12±0.8 grams inleptin-administered mice, P=NS for both parameters, (FIG. 3).Macroscopically, tumors in SCID beige mice, in leptin andsaline-administered mice, featured no central areas of necrosis.

Example 9

In-vitro Effect of Leptin Administration on Natural Killer CellCytotoxicity

Leptin manifested a dose-dependent increase in natural killer cellcytotoxicity in vitro (FIG. 10). A 4 hour incubation of mouse-derivednatural killer cells with YAC cells in the presence of increasing dosesof mouse leptin (0.01 mcg/ml to 1 mcg/ml) resulted in a significantlyincreased natural-killer cell mediated lysis of YAC cells (manifested asincreased LDH release). This leptin-medicated dose-response enhancementin cytotoxicity was notable in each of the tested NK-YAC ratios (5:1 to0.04:1 NK-YAC ratios). In each NK-YAC ratio, cytotoxicity increased withincreasing doses of leptin. In the highest tested NK-YAC ratio of 5:1,administration of 1 mcg/ml leptin resulted in a 100% 4-hourcytotoxicity.

Example 10

In-vitro Effect of Leptin Administration on Natural Killer CellProliferation

High dose leptin (1 mcg/ml) but not lower doses (0.1 & 0.01 mcg/ml)induced in vitro proliferation of natural killer cell. Incubation of1×10⁴ natural killer cells with 0, 0.01, and 0.1 mcg/ml mouse leptinresulted in thymidine uptake of 1361±143, 1302±173, and 1359±229 CPM,respectively, P=NS. Incubation with 1 mcg/ml leptin, on the other hand,resulted in a significantly elevated thymidine uptake of 2733±52 CPM.P<0.05

Example 11

In-vitro Effect of Leptin Administration on Hepatocellular CarcinomaCell Proliferation

In-vitro administration of leptin to HEPA 3B hepatocellular carcinomacell culture resulted in a dose-dependent inhibition of tumor cellgrowth (FIG. 11). A 5-day incubation of 10³ HCC cells in the presence ofincreasing leptin doses resulted in significant reduction in thymidineincorporation, from 7678±2603CPM in the absence of leptin to 3371±1178CPM in the presence of 0.1 mcg/ml leptin, and 832±289 CPM in thepresence of 1 mcg/ml leptin, P<0.001. Maximal inhibition was noted at0.1 mcg/ml, with no added inhibition noted with the addition of 1 mcg/mlleptin (1287±412) as compared to 0.1 mcg/ml leptin (P=NS).

Example 12

In-vitro Effect of Leptin Administration Hepatocellular CarcinomaProliferation in Presence of Natural Killer Cells

The NK cell mediated effect of leptin on HCC cell proliferation wasassessed by incubation of 10³ HCC cells for 5 days in the presence of10⁴ irradiated natural killer cells in the presence of increasing leptindoses. Incubation of HCC cells in the presence of natural killer cellsresulted in further inhibition of tumor cell growth, from 885±152 CPMwithout leptin, to 815±117 CPM, 724±44, & 613±95 CPM in the presence of0.01, 0.1, and 1 mcg/ml leptin, respectively, (P<0.05 for 0.1 & 0.01mcg/ml leptin).

Example 13

In-vitro Effect of Leptin Administration on Hepatoma Cell mRNAExpression

HEPA 3B cells expressed leptin receptor mRNA. To determine possiblemechanisms by which leptin mediates its inhibitory affect on HCC cellgrowth, Hep3B cells were incubated in the presence of increasing leptindoses (0.5 mcg/ml, 5 mcg/ml, 50 mcg/ml for 45 minutes, 2 and 6 hours.Semi quantitative rtPCR demonstrated that leptin administration, even atthe lowest doses, resulted in increased mRNA expression of STAT2 andSOCS1 in HCC cells (FIG. 12). Increased mRNA expression was notableafter two hours for STAT2 and SOCS1. No effect of leptin administrationon mRNA expression was noted for STAT 1 & 3-6, SOCS 2-4, and CIS.

The invention claimed is:
 1. A method of treating cancer or an immunedisorder caused by immune suppression in a subject comprisingadministration of a reagent that increases the activity of leptin insaid subject wherein a shift takes place in the Th1/Th2 balance towardsa pro-inflammatory state, wherein said cancer or said immune disorder isHepatocellular carcinoma (HCC).
 2. A method for the treatment of animmune-related disorder in a subject comprising increasing the activityof leptin in said subject by administering to said subject animmunomodulatory amount of leptin, any functional homologue, functionalderivative, or functional fragment of leptin, or any combination of anyof the foregoing, wherein said immune-related disorder is Hepatocellularcarcinoma (HCC).
 3. The method of claim 1 wherein increasing theactivity in said subject comprises raising the amount of leptin in saidsubject.
 4. The method of claim 3 wherein said increase in leptin amountis caused by administering to said subject an immunomodulatory amount ofleptin, any functional homologue, functional derivative, or functionalfragment of leptin, or any combination of any of the foregoing.